Reagents and methods for sequencing

ABSTRACT

The disclosure provides a plurality of nucleic acid sequences comprising multiple variants of a reference sequence. The disclosure further provides plasmids, cells, methods and kits comprising the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. application No. 61/903,268, filed Nov. 12, 2013, which disclosure is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

A significant challenge facing testing laboratories is quality control. Some reports have indicated that mutations in cancer genes were correctly identified by only 70% of testing laboratories (Bellon, et al. External Quality Assessment for KRAS Testing Is Needed: Setup of a European Program and Report of the First Joined Regional Quality Assessment Rounds. Oncologist. 2011 April; 16(4): 467-478). Questions have been raised regarding how to monitor next generation sequencing and assays as well as the concordance of variant calls across multiple platforms, library preparation methods, and bioinformatic pipelines. Compositions and methods providing a flexible, single reagent representing a large number of genetic variants are desired by those of ordinary skill in the art and are described herein.

SUMMARY OF THE INVENTION

The disclosure provides compositions, controls, plasmids, cells, methods and kits comprising nucleic acid molecules.

In one embodiment, a nucleic acid molecule comprising multiple variants of a reference is disclosed. In other embodiments, a mixture or combination of nucleic acid molecules comprising variants of the reference sequence are disclosed.

In certain embodiments, the nucleic acid molecule or mixture of nucleic acid molecules comprise one or more variants present at a high or low-frequency.

In certain embodiments, the disclosure provides a control reagent comprising multiple nucleic acid molecules.

In yet another embodiment, a kit comprising at least one nucleic acid molecule or mixture of nucleic acid molecules comprising variants is disclosed

In another embodiment, a method for confirming the validity of a sequencing reaction is disclosed. The method comprises including a known number of representative sequences and/or variants thereof in a mixture comprising a test sample potentially comprising a test nucleic acid sequence, and sequencing the nucleic acids in the mixture, wherein detection of all of the representative sequences and/or variants in the mixture indicates the sequencing reaction was accurate.

The disclosure also provides a composition comprising multiple nucleic acid species wherein the nucleic acid sequence of each species differs from its neighbor species by a predetermined percentage.

In certain embodiments, a method is provided that comprises sequencing a nucleic acid species in order to calibrate a sequencing instrument.

In yet other embodiments, the disclosure provides plasmids and cells encoding the nucleic acids or mixture of nucleic acids disclosed herein.

The disclosure also provides a plasmid and/or a cell comprising multiple nucleic acid species wherein the nucleic acid sequence of each species differs from its neighbor species by a predetermined percentage.

The disclosure further provides a frequency ladder. The frequency ladder comprises a plurality of variants at different frequencies.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Exemplary EGFR amplicon selection.

FIG. 2 is a graph showing variant frequency at each nucleotide position as well as percentage A and G content. Sequences 1-5 are the same and are used to dilute out sequences 6-10. Each sequence is found in its own cassette, and all cassettes are found in the same plasmid. This design provides an absolute truth—e.g., there is 10% sequence 6 in this design. In contrast to mixing with genomic sequence, this provides the most precision when making a 10% mix. This could be used to calibrate assays.

FIG. 3 is a schematic of an exemplary plasmid with 10 sequences and restriction sites, leading to equal ratios of each sequence.

FIG. 4 is a graph showing the frequency percentage per run comprising Panel A (FLT3, PDGFRA, FGFR3, CSF1R, EGFR, HRAS, and TP53).

FIG. 5 is a graph showing the frequency percentage per run of Panel A and Panel B (TP53, PIK3CA, GNA11, VHL, FBXW7, RET, HNF1A, and STK11)

FIG. 6 is a graph showing the frequency percentage per run of Panel A, Panel B, and Panel C (RB1, EGFR, ABL1, ERBB2, and ATM).

FIG. 7 is a graph showing the frequency percentage per run of Panel A, Panel B, Panel C, and Line D, which represents the number of reads (i.e., coverage)

FIG. 8 is a graph showing the number of variants (deletions, insertions, complex, multiple nucleotide variants (MNV), and single nucleotide variants (SNV)) and average number of variants detected across multiple sites using CHPv2 (AMPLISEQ™ Cancer Hotspot Panel version 2), TSACP (TRUSEQ™ Amplicon Cancer Panel), and TSTP (TRUSIGHT™ Tumor Panel).

FIG. 9 is a graph showing analysis conducted with data from sites that tested two lots of the control at least once or one lot at least twice. Detection is indicated in dark squares and absence light squares.

FIG. 10 is a graph showing the mean number and mean percentage SNPs detected for CHPv2 and TTP.

FIG. 11 is a graph showing the mean number and mean percentage of SNPs detected for CHPv2 and TACP.

DETAILED DESCRIPTION OF THE INVENTION

Provided herein are compositions, methods, kits, plasmids, and cells comprising nucleic acid reference sequences and variants of a reference sequence. The compositions disclosed herein have a variety of uses, including but not limited to, assay optimization, validation, and calibration; peer-to-peer comparison; training and PT/EQA, QC monitoring, reagent QC, and system installation assessment.

There is a recognized need in the market for flexible, reliable control materials for NGS testing (see Assuring the Next Quality of Next-Generation Sequencing in Clinical Laboratory Practice; Next Generation Sequencing: Standardization of Clinical Testing (Nex-SToCT) Working group Principles and Guidelines, Nature Biotechnology, doi:10.1038/nbt.2403; and ACMG Clinical laboratory standards for next generation sequencing, American College of Medical Genetics and Genomics, doi: 10.1038/gim.2013.92). This disclosure provides such control materials.

This disclosure relates to control reagents representing reference sequences and/or variants thereof (e.g., mutations) that may be used for various purposes such as, for instance, assay validation/quality control in sequencing reactions (e.g., next generation sequencing (NGS) assays). Traditional metrics used to characterize the quality of a sequencing reaction include, for instance, read length, minimum quality scores, percent target-mapped reads, percent pathogen-specific reads, percent unique reads, coverage levels, uniformity, percent of non-covered targeted bases and/or real-time error rate. Parameters that may affect quality include, for instance, the types and/or number of analytes being monitored (e.g., the types and number of polymorphisms (single or multiple nucleotide polymorphisms (SNPs, MNPs)), insertions and/or deletions, amplicons, assay contexts and/or limits of detection), sample type (e.g., mammalian cells, infectious organism, sample source), commutability (e.g., validation across multiple technology platforms and/or types of screening panels being utilized), sample preparation (e.g., library preparation type/quality and/or type of sequencing reaction (e.g., run conditions, sequence context)), and/or other parameters. Those of ordinary skill in the art realize, for instance, that the quality of such reactions may vary between laboratories due to subtle differences in guidelines, the metrics and parameters mentioned above, the reference standards used, and the fact that many NGS technologies are highly complex and evolving. This disclosure provides quality control reagents that may be used in different laboratories, under different conditions, with different types of samples, and/or across various technology platforms to confirm that that assays are being carried out correctly and that results from different laboratories may be reliably compared to one another (e.g., that each is of suitable quality). In some embodiments, the problem of confirming the quality of a sequencing reaction is solved using a multiplex control comprising multiple nucleic acid fragments, each representing a different variant of a reference sequence.

In certain embodiments, a control reagent for use in sequencing reactions is provided. The control reagent may comprise one or more components that may be used alone or combined to assess the quality of a particular reaction. For instance, some assays are carried out to identify genetic variants present within a biological sample. The control reagents described herein may also provide users with the ability to compare results between laboratories, across technology platforms, and/or with different sample types. For instance, in some embodiments, the control reagent may represent a large number of low percentage (e.g., low frequency) variants of different cancer-related genes that could be used to detect many low percentage variants in a single assay and/or confirm the reliability of an assay. The control reagent could be used to generate numerous data points to compare reactions (e.g., run-to-run comparisons). The control reagent may be used to determine the reproducibility of variant detection over time across multiple variables. The control reagent may be used to assess the quality of a sequencing run (i.e., that the instrument has sufficient sensitivity to detect the included variants at the given frequencies). The control reagent may also be used to differentiate between a proficient and a non-proficient user by comparing their sequencing runs, and/or to differentiate the quality of reagents between different lots. The control reagent may also aid in assay validation studies, as many variants are combined in one sample material. This obviates the need for multiple samples containing one or two variants each, and greatly shortens the work and time required to validate the assay.

The control reagent typically comprises one or more nucleic acid (e.g., DNA, RNA, circular RNA, hairpin DNA and/or RNA) fragments containing a defined reference sequence of a reference genome (defined as chromosome and nucleotide range) and/or one or more variants of the reference sequence. The source material for the variants may be genomic DNA, synthetic DNA, and combinations thereof. A variant typically includes nucleotide sequence variations relative to the reference sequence. The variant and reference sequence typically share at least 50% or about 75-100% (e.g., any of about 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%) sequence identity. In some embodiments, however, the identity shared may be significantly less where, for example, the variant represents a deletion or insertion mutation (either of which may be up to several kilobases or more). An exemplary deletion may be, for instance, recurrent 3.8 kb deletion involving exons 17a and 17b within the CFTR gene as described by Tang, et al. (J. Cystic Fibrosis, 12(3): 290-294 (2013) (describing a c.2988+1616_c.3367+356del3796ins62 change, flanked by a pair of perfectly inverted repeats of 32 nucleotides)). In some embodiments, variants may include at least one of a single nucleotide polymorphism (SNP), one or more multiple nucleotide polymorphism(s) (MNV), insertion(s), deletion(s), copy number variation(s), gene fusion(s), duplication(s), inversion(s), repeat polymorphism(s), homopolymer(s), non-human sequence(s), or any combination thereof. Such variants (which may include by reference any combinations) may be included in a control reagent as part of the same or different components. The reference sequence(s) and/or variants may be arranged within a control reagent as cassettes.

Cassettes contains a reference sequence or variant adjoined and/or operably linked to one or more restriction enzyme site(s), sequencing primer(s) site, and/or hairpin-forming site(s). In some embodiments, it may be useful to include different types of sequences adjacent to each cassette; for instance, it may be useful to design one cassette to be adjacent to a restriction enzyme and/or a hairpin sequence. Doing so may help prevent problems such as cross-amplification between adjacent fragments/cassettes. As such, each reference sequence and/or variant may be releasable and/or detectable separate from any other reference sequence and/or variant. The typical cassette may be about 400 bp in length but may vary between 50-20,000 bp (e.g., such as about any of 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 800, 900, 1000, 2500, 5000, 7500, 10000, 12500, 15000, 17500, or 20000 bp). Each control reagent may comprise one or more cassettes, each representing one or more reference sequence(s) and/or variant(s) (e.g., each being referred to as a “control sequence”). Each reference sequence and/or variant may be present in a control sequence and/or control reagent at percentage of about any of 0.1% to 100% (e.g., about any 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2.5, 5, 7.5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%). For instance, a control sequence or control reagent that is 100% reference sequence or variant would be a reagent representing only one reference sequence or variant. Similarly, a control sequence or control reagent comprising 50% of a variant would be a control reagent representing only up to two reference sequences and/or variants. The remaining percentage could consist of other sequences such as control sequences and the like.

In certain embodiments a T7 or other promoter can be present upstream of each cassette. This allows for massively parallel transcription of many gene regions. This technique facilitates construction of a control containing equivalent amounts of each target sequence. When there are equivalent amounts of many targets, ease of use of the control is increased. For example, contamination in a control in a patient sample would be easier to detect because all transcripts would show up in the contaminated sample. It is highly unlikely for patient samples to contain, e.g., a large number of fusion transcripts, such an assay result would signal the user that that a contamination issue is present. This is in contrast to a situation in which only one transcript is present at a much higher abundance in a contaminated sample—which could lead to the contaminant being mistaken for a true positive signal. The ability to construct a control with equivalent amounts of each target sequence eliminates the potential for this type of error.

In certain embodiments, the reference sequence(s) and/or variants may be adjoined and/or operably linked to one or more different restriction enzyme sites, sequencing primer site(s), and/or hairpin-forming sites. As described above, certain designs may be used to prevent problems such as cross-amplification between reference sequences and/or variants. In some embodiments, the control sequences and/or cassettes may optionally be arranged such that the same are releasable from the control reagent. This may be accomplished by, for instance, including restriction enzyme (RE) sites at either end of the control sequence. A control reagent may therefore be arranged as follows: RE site/control sequence/RE site. The RE sites may be the same and/or different from one another. The RE sites in one cassette may also be the same and/or different to those present in any other cassette. As such, the control sequences may be released from the cassette as desired by the user by treating the control sequence with one or more particular restriction enzymes.

In some embodiments, the control reagent may comprise multiple components that may be used together. In certain embodiments, the multiple components comprise a first and a second component which may be plasmids comprising different control sequences and/or different arrangements of the same control sequences. Thus, the components may represent the same or different reference sequences and/or variants. Such components may be used together as a panel, for instance, such that a variety of reference sequences and/or variants may be assayed together. Where the reference sequences and/or variants are the same, each component may include those variants in different cassette arrangements and/or forms. In some embodiments, the multiple components may comprise a first component representing one or more SNP variants and a second component representing one or more multiple nucleotide polymorphism(s), insertion(s), deletion(s), copy number variation(s), gene fusion(s), duplication(s), inversion(s), repeat polymorphism(s), homopolymer(s), and/or non-human sequence(s). The components may be the same or different types of nucleic acids such as plasmids, with each comprising the same or different variants of one or more reference sequences arranged as described herein or as may be otherwise determined to be appropriate by one of ordinary skill in the art. In some embodiments, different types of plasmids may be combined to provide a multi-component control reagent representing many different reference sequences and/or variants.

Plasmids can be quantified by any known means. In one embodiment, quantitation of each plasmid is performed using a non-human ‘xeno’ digital PCR target sequence. The exact copy number of the plasmid is determined. The exact copy number of genomic DNA is also determined (obtained by quantification of genomic target site(s)). With this information, controls can be accurately and reproducibly developed that contain all targets/variants within a tight frequency range.

The variants may be contained within the control reagent as DNA fragments, each containing a defined sequence derived from a reference genome (defined as chromosome and nucleotide range) with one or more variations (e.g., nucleotide differences) introduced into the fragment. A variant may be, for instance, a sequence having one or more nucleotide sequence differences from the defined sequence (e.g., a reference sequence). For instance, an exemplary reference sequence may comprise “hotspots” suitable for modification. Such hotspots may represent nucleotides and/or positions in a reference sequence that occur in nature (e.g., mutations observed in cancer cells). One or more of such hotspots may be modified by changing one or more nucleotides therein to produce a control sequence (or portion thereof) that may be incorporated into a control reagent. For example, modification of the exemplary epidermal growth factor receptor (EGFR) Ex 19 reference sequence to produce control sequences (Hotspots 1, 2, 3, 4, 5) is shown below (see also, FIG. 1):

Wild Type (e.g., EGFR Ex19) (SEQ ID NO: 1) CCAAGCTC  (SEQ ID NO: 2) AGGATCTTGA  (SEQ ID NO: 3) AACTGAATTC  (SEQ ID NO: 4) AAAAAG  (SEQ ID NO: 5) ATCAAAGTGC (400 bp) Hotspot ID 1 (SEQ ID NO: 6) CCAA T CTC  (SEQ ID NO: 2) AGGATCTTGA  (SEQ ID NO: 3) AACTGAATTC  (SEQ ID NO: 4) AAAAG  (SEQ ID NO: 5) ATCAAAGTGC 

Control Sequence Contains Multiple Hotspots CCAATCTC (SEQ ID NO: 6; HOTSPOT ID 1) . . . AGGAACTTGA (SEQ ID NO: 7; HOTSPOT ID 2) . . . AACTCAATTC (SEQ ID NO: 8; HOTSPOT ID 3) . . . ATAAAG (SEQ ID NO: 9; HOTSPOT ID 4) . . . ATGAAAGTGC (SEQ ID NO: 10; HOTSPOT ID 5). This exemplary control sequence thereby represents multiple EGFR variants (e.g., Hotspot IDs 1, 2, 3, 4, 5, etc.) A control reagent may comprise multiple control sequences, each representing one or more variants of the same or different reference sequences. Any number of variants may be represented by a control sequence, and any number of control sequences may be included in a control reagent. A control reagent may comprise, for instance, a number of variants such that the all possible variants of a particular reference sequence are represented by a single control reagent. For instance, the control reagent may comprise multiple SNPs, MNPs, deletions, insertions and the like, each representing a different variant of the reference sequence. Additional, exemplary, non-limiting variants are shown in Tables 1A and 1B and Table 6.

Control reagents may also be designed to represent multiple types of control sequences. For instance, control reagents may be designed that represent multiple types of reference sequences and/or variants thereof (which may be found in control sequences alone or in combination). Exemplary categories of control sequences for which the control reagents described herein could have relevance include not only the aforementioned cancer-related areas but also fields of inherited disease, microbiology (e.g., with respect to antibiotic resistance mutations, immune-escape related mutations), agriculture (e.g., plant microbe and/or drug resistance-related mutations), livestock (e.g., mutations related to particular livestock traits), food and water testing, and other areas. Exemplary combinations (e.g., panels) of cancer-related reference sequences that may be represented by a particular control reagent (or combinations thereof) are shown in Table 2.

The control reagents and methods for using the same described herein may provide consistent control materials for training, proficiency testing and quality control monitoring. For instance, the control reagents may be used to confirm that an assay is functioning properly by including a specific number of representative sequences and/or variants thereof that should be detected in an assay and then calculating the number that were actually detected. This is exemplified by the data presented in Table 3:

As illustrated in Table 3, a “bad run” is identified where the number of variants detected does not match the number of variants expected to be detected (e.g., included in the assay). As shown in the exemplary assay of Table 3, if a particular control reagent (or combination thereof) used in an assay includes 15 representative sequences and/or variants thereof, all 15 should be detected if the assay is properly carried out. If less than 15 of these control sequences are not detected, the assay is identified as inaccurate (e.g., a “Bad Run”). If all 15 of the sequences are detected, the assay is identified as accurate (e.g., a “Good Run”). Variations of this concept are also contemplated herein, as would be understood by those of ordinary skill in the art.

In certain embodiments, the control reagent may be prepared by mixing variant DNA fragments (e.g., as may be incorporated into a plasmid) with genomic DNA or synthesized DNA comprising “wild-type” (e.g., non-variant) sequence. Such sequence may be obtained from or present in control cells (e.g., naturally occurring or engineered/cultured cell lines). In some embodiments, the wild-type sequence may be included on a DNA fragment along with the variant sequence, or the variant sequences may be transfected into and/or mixed with cells (e.g., control cells). In certain embodiments, such mixtures may be used to prepare formalin-fixed, paraffin-embedded (FFPE) samples (e.g., control FFPE samples), for example. For instance, in some embodiments, the control reagent may be prepared and tested by designing a control sequence (e.g., an amplicon) comprising a representative sequence and/or variant thereof; designing restriction sites to surround each amplicon; synthesizing a nucleic acid molecule comprising a cassette comprising the amplicon and the restriction sites; and, incorporating the cassette into a plasmid backbone. The construct may then be tested by sequencing it alone (e.g, providing an expected frequency of 100%) or after mixing the same with, for example, genomic DNA at particular expected frequencies (e.g., 50%). Such constructs may also be mixed with cells for various uses, including as FFPE controls.

In certain embodiments, the control reagents described herein can also be used to provide a frequency ladder. A frequency ladder is composed of many variants at different frequencies. In some embodiments, the control reagent could be used to provide an “ladder” in, for example, 5% increments of abundance (e.g., about any of 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% abundance). For example, the ladder could be constructed by taking a single sample with many different variants present at high (e.g., 80% allele frequency) and making dilutions down to low frequencies. Alternatively, the ladder could be a single sample containing variants at different frequencies. The ladder could be used as a reference for many sample types, including somatic variants at low abundance (e.g., tumor single nucleotide polymorphisms), or germline variants present at, as a non-limiting example, about 50% abundance. Such a ladder may also be used to determine instrument limits of detection for many different variants at the same time. This saves users time in finding materials containing one to a few variants and resources for testing because all variants are present in a single sample rather than many. An example is provided in Table 8:

As shown in Table 8, a ladder was constructed by diluting a sample containing 555 variants starting at approximately 50% frequency down to ˜3% frequency. The ladder was tested in duplicate using the Ion AMPLISEQ® Cancer Hotspot Panel v2 using the Ion Torrent PERSONAL GENOME MACHINE® (PGM). The frequencies for 35 of the variants are reported for each sample tested. The shaded cells indicate that the variant was not detected. Such data could be used to establish the limit of detection for each variant.

The ladder could be used across many platforms, including Sanger sequencing and next generation platforms, and both RUO and IVD applications could benefit from use of this standard. The frequency ladder could also serve as internal controls in sequencing reactions, much like the 1 kb DNA ladder serves as a reference in almost every agarose gel. As an example, one design would provide five unique and five identical sequences as shown in FIGS. 2 and 3. As shown therein, in one sequence position, there is a variant present in only one of these ten sequences. At a second position, the variant is present in two of the ten sequences. At the third position, the variant is present in three of the sequences, and so on. This would yield variants at 10% frequency increments from 0-100%. This is a simplified example and random intervening sequences may be necessary to prevent sequencing artifacts. The product could take any suitable form such as an oligonucleotide (e.g., PCR fragment or synthetic oligonucleotide), plasmids, or one plasmid with concatenated sequences separated by identical restriction enzyme sites (FIG. 3). An advantage of having all sequence variants on one plasmid is that the relative levels of all ten sequences within a mixture would be well controlled; during manufacturing, the plasmid could be cleaved between each sequence with the same enzyme, giving rise to ten fragments at equal ratios. As would be understood by those of ordinary skill in the art, derivations of such a ladder could include different variant types (e.g., insertions and/or deletions), every nucleotide change could be incorporated into the design (e.g., A−>C, A−>T, etc.), and/or smaller increments could provide fine-tuned measurement at abundances lower than 10%. For instance, a second plasmid with other mutations could be added to the initial plasmid at a one to nine ratio, yielding variants at even lower frequencies. Such a low frequency sequencing ladder could be an essential control when measuring somatic mutations that appear, for example, at <10% abundance. In some embodiments, such a sequencing ladder may comprise multiple nucleic acid species wherein the nucleic acid sequence of each species differs from its neighbor species by a predetermined percentage (e.g., about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10% or more). Each species may comprise, for instance, any suitable number of nucleotides (e.g., about any of 5, 10, 20, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500). Each species may also comprise a homopolymer sequence of at least 3 nucleotides. In some embodiments, the nucleic acid of the species is DNA, and these may be encoded on vectors such as plasmids and/or by and/or within cells. In some embodiments, each species may comprise a nucleic acid bar code that may be unique to each species. Methods comprising sequencing the nucleic acid species to calibrate a sequencing instrument, to obtain data for sequencing instrument development work, including algorithm development, base calling, variant calling, and/or verify that an instrument is functioning properly (e.g., IQ/OQ/PQ) are also contemplated.

One of ordinary skill in the art would understand that the control reagents described herein are broadly useful in a variety of sequencing systems and/or platforms. For instance, the control reagents described herein may be used in any type of sequencing procedure including but not limited to Ion Torrent semiconductor sequencing, IIlumina MISEQ®, capillary electrophoresis, microsphere-based systems (e.g., Luminex), Roche 454 system, DNA replication-based systems (e.g., SMRT by Pacific Biosciences), nanoball- and/or probe-anchor ligation-based systems (Complete Genomics), nanopore-based systems and/or any other suitable system.

One of ordinary skill in the art would also understand that the control reagents described herein are broadly useful in a variety of nucleic acid amplification-based systems and/or platforms. The control reagents described herein may used in and/or with any in vitro system for multiplying the copies of a target sequence of nucleic acid, as may be ascertained by one of ordinary skill in the art. Such systems may include, for instance, linear, logarithmic, and/or any other amplification method including both polymerase-mediated amplification reactions (such as polymerase chain reaction (PCR), helicase-dependent amplification (HDA), recombinase-polymerase amplification (RPA), and rolling chain amplification (RCA)), as well as ligase-mediated amplification reactions (such as ligase detection reaction (LDR), ligase chain reaction (LCR), and gap-versions of each), and combinations of nucleic acid amplification reactions such as LDR and PCR (see, for example, U.S. Pat. No. 6,797,470). Such systems and/or platforms may therefore include, for instance, PCR (U.S. Pat. Nos. 4,683,202; 4,683,195; 4,965,188; and/or 5,035,996), isothermal procedures (using one or more RNA polymerases (see, e.g., PCT Publication No. WO 2006/081222)), strand displacement (see, e.g., U.S. Pat. No. RE39007E), partial destruction of primer molecules (see, e.g., PCT Publication No. WO 2006/087574)), ligase chain reaction (LCR) (see, e.g., Wu, et al., Genomics 4: 560-569 (1990)), and/or Barany, et al. Proc. Natl. Acad. Sci. USA 88:189-193 (1991)), Qβ RNA replicase systems (see, e.g., PCT Publication No. WO 1994/016108), RNA transcription-based systems (e.g., TAS, 3SR), rolling circle amplification (RCA) (see, e.g., U.S. Pat. No. 5,854,033; U.S. Patent Application Publication No. 2004/265897; Lizardi et al. Nat. Genet. 19: 225-232 (1998); and/or Bailer et al. Nucleic Acid Res., 26: 5073-5078 (1998)), and/or strand displacement amplification (SDA) (Little, et al. Clin. Chem. 45:777-784 (1999)), among others. These systems, along with the many other systems available to the skilled artisan, may be suitable for use with the control reagents described herein.

In one embodiment, a control reagent may be designed and tested using one or more of the steps below:

-   -   designing a control sequence (e.g., an amplicon) comprising a         representative sequence of a particular gene of interest and/or         variants thereof (e.g., those targeted by commercially-available         NGS tests such as the AMPLISEQ Cancer Hotspot Panel v2, and/or         the TRUSEQ Amplicon Cancer Panel);     -   identifying sequence from a genome reference source (e.g.,         Genome Reference Consortium Human Reference 37 (GRCh37))         encompassing the amplicon;     -   designing a cassette comprising an ˜400 bp sequence comprising         the amplicon surrounded by (e.g., 5′ and 3′) the genomic         sequence identified in step b);     -   designing restriction sites to surround each cassette prepared         in step c) (e.g., where one version may additionally include         sequences that create a hairpin when the DNA is         single-stranded);     -   synthesizing a nucleic acid molecule comprising the cassette of         step c) and restriction sites of step d) using a common vector         (e.g., pUC57) (e.g., “plasmid V1”);     -   preparing a second plasmid (e.g., “plasmid V2”) comprising         multiple fragments of the gene of interest (and/or variants         thereof) with a hairpin structure and a restriction site between         each region;     -   optionally, linearizing the variant sequences contained within         plasmids V1 and/or V2 with a restriction enzyme;     -   mixing the variants with genomic DNA (e.g., wild-type gDNA) at a         particular expected variant frequency (e.g., approximately 50%);     -   optionally, testing the “variant sequence” alone (e.g.,         providing an expected variant frequency of 100%);     -   performing variant detection using NGS.

In certain embodiment, individual cassettes can be synthesized for all genes of interest and combined with wild type. In certain embodiments, a cassette can be designed with a plurality of variants, which do not interfere with the detection of variants near or adjacent thereto.

In some embodiments, NGS may be performed using the Ion Personal Genome Machine (PGM) by first constructing libraries following the user manuals for the Ion AMPLISEQ® Library Preparation Manual with AMPLISEQ® Cancer Hotspot Panel v2 reagents; preparing template-positive Ion sphere particles (ISPs) and enriching the same using the Ion OneTouch2 instrument following the Ion PGM Template OT2 200 Kit Manual; sequencing using the Ion PGM Sequencing 200 Kit v2 Manual or Sequencing on the Illumina MISEQ® following the TRUSEQ® Amplicon Cancer Panel user manual or the Illumina MiSeq® user manual; and, performing data analysis for PGM using the Torrent Variant Caller v3.4 and v3.6, and for MISEQ® using the MISEQ® Reporter v2.3).

The reagents and methods described herein may be used in a variety of settings with a variety of samples. For instance, these reagents and methods may be used to analyze biological samples such as serum, whole blood, saliva, tissue, urine, dried blood on filter paper (e.g, for newborn screening), nasal samples, stool samples or the like obtained from a patient and/or preparations thereof (e.g., FFPE preparations). In some embodiments, control preparations comprising the control reagents described herein may be provided.

This disclosure further relates to kits comprising one or more control reagents described herein. The kits may be used to carry out the methods described herein or others available to those of ordinary skill in the art along with, optionally, instructions for use. A kit may include, for instance, control sequence(s) including multiple reference sequences and/or variations thereof in the form of, for instance, one or more plasmids. In some embodiments, the kit may contain a combination of control sequences organized to provide controls for many variations of one or more reference sequences. In some embodiments, the variations may relate to an oncogene that is diagnostic for a particular cancer. In some embodiments, for instance, the kit may comprise control reagents and/or control samples (e.g., tissue samples) known to cover the breadth of mutations known for a particular cancer. In some embodiments, the variations of the marker are variations of a mutation in a gene that are prognostic for the usefulness of treating with a drug. In some embodiments, the marker or markers are for a particular disease and/or a variety of diseases (e.g., cancer, infectious disease). In some embodiments, the control reagent(s) may be included in a test to ascertain the efficacy of a drug in testing for the presence of a disease and/or progression thereof. In some embodiments, the kit may comprise control reagents for testing for a series of diseases that have common characteristics and/or symptoms (e.g., related diseases). In some embodiments, the marker may have unknown significance but may otherwise be of interest to the user (e.g., for basic research purposes). The kit may also include a container (e.g., vial, test tube, flask, bottle, syringe or other packaging system (e.g., include injection or blow-molded plastic containers) into which one or more control reagents may be placed/contained, and in some embodiments, aliquoted). Where more than one component is included in the kit, it will generally include at least one second, third or other additional container into which the additional components can be separately placed. Various combinations of components may also be packaged in a single container. The kits may also include reagent containers in close confinement for commercial sale. When the components of the kit are provided in one and/or more liquid solutions, the liquid solution comprises an aqueous solution that may be a sterile aqueous solution. As mentioned above, the kit may also include instructions for employing the kit components as well as the use of any other reagent not included in the kit. Instructions may include variations that may optionally be implemented. The instructions may be provided as a separate part of the kit (e.g., a paper or plastic insert or attachment) or as an internet-based application. In some embodiments, the kit may control reagents relating to between any number of reference sequences and/or variants thereof which may be detected alone or in combination with one another (e.g., a multiplex assay). In some embodiments, the kit may also comprise at least one other sample containing a defined amount of control reagent and “control” test cell admixed such that the same may provide a reference point for the user. Kits may further comprise one or more of a polymerase and/or one or more oligonucleotide primers. Other variations and arrangements for the kits of this disclosure are contemplated as would be understood by those of ordinary skill in the art.

Thus, in some embodiments, the disclosure provides a nucleic acid molecule or mixture of nucleic acid molecules comprising multiple variants of a reference sequence, each variant sequence may optionally be releasable from the nucleic acid molecule. In certain embodiments, the nucleic acid molecule or mixture of nucleic acid molecules comprises variants releasable from the nucleic acid molecule using a restriction enzyme.

In some embodiments, the nucleic acid molecule or mixture of nucleic acid molecules comprises at least one single nucleotide polymorphism (SNP), multiple nucleotide polymorphisms (MNP), insertion, deletion, copy number variation, gene fusion, duplication, inversion, repeat polymorphism, homopolymer of a reference sequence, and/or a non-human sequence. In some embodiments, the nucleic acid molecule or mixture of nucleic acid molecules comprises at least 5 variants. In certain embodiments, at least 15, 20, 30, 50, 100, 200, 300 400, 700, 1000 variants are present. In yet other embodiments, greater than 1000 variants are present. In some embodiments, each variant is present (e.g., in the sample being tested) at a high or low-frequency. For instance, in certain embodiments, each variant may be present at a frequency of 1%, 5%, 10%, 15%, 20%, 30%, 40% or 50% or more. In other embodiments, each variant may be present at a frequency of less than 50%, less than 40%, less than 20%, less than 15%, less than 10%, less than 5%, less than 3%, less than 1%, less than 0.5%, less than 0.1%, and any integer in between.

An advantage of the disclosed control materials is that the “truth” of a sample is known. There are currently no reference materials for which absolute frequency (i.e, the truth) is known, that is, the actual frequency of a given variant or combination of variants present are not known. In contrast, in the disclosed control materials, the actual frequency of variants is known.

Attendant to the teachings of this disclosure, standardized control materials for next generation sequencing (NGS) assays can be produced. Issues such as variant call differences between sites, variability of reagents across instruments, variation introduced by diverse bioinformatics pipelines and filters, run-to-run and lab-to-lab variability can be identified and resolved and/or obviated utilizing the control materials.

A further advantage is that the control materials disclosed herein can comprise any number and type of variants, including insertions and deletions of differing lengths, large numbers of SNPs, etc. No other control material exists that provide such diversity.

The variants can be any of interest. There is no limit provided herein with respect to the type and number of variants that can be utilized in the current disclosure.

In certain embodiments, modified nucleotides can be utilized as variants. In certain embodiments, methylation can be detected. For example, CpG methylation can be utilized as a biomarker variant.

This disclosure also provides reagents and methods for confirming the validity of a sequencing reaction by including a known number of representative sequences and/or variants thereof in a mixture comprising a test sample potentially comprising a test nucleic acid sequence and sequencing the nucleic acids in the mixture, wherein detection of all of the representative sequences and/or variants in the mixture indicates the sequencing reaction was accurate. The representative sequences and/or variants may be of the type described herein. Compositions comprising the same are also provided. The pre-determined percentage may be, for instance, about 1, 5 or 10%. And each species may be from, for instance, 20-500 nucleotides. Each species may comprise a homopolymer sequence of at least 3 nucleotides. The nucleic acids may be DNA. Each species may possess a nucleic acid barcode that may be unique to each species. The nucleic acid species described herein may be used to calibrate a sequencing instrument, for instance. Kits comprising such species, optionally further comprising one or more polymerases and/or one or more oligonucleotide primers are also provided. Plasmids and/or cells comprising multiple nucleic acid species wherein the nucleic acid sequence of each species differs from its neighbor species by a predetermined percentage are also provided.

It is to be understood that the descriptions of this disclosure are exemplary and explanatory only and are not intended to limit the scope of the current teachings. In this application, the use of the singular includes the plural unless specifically stated otherwise. Also, the use of “comprise”, “contain”, and “include”, or modifications of those root words, for example but not limited to, “comprises”, “contained”, and “including”, are not intended to be limiting. Use of “or” means “and/or” unless stated otherwise. The term “and/or” means that the terms before and after can be taken together or separately. For illustration purposes, but not as a limitation, “X and/or Y” can mean “X” or “Y” or “X and Y”. Whenever a range of values is provided herein, the range is meant to include the starting value and the ending value and any value or value range therebetween unless otherwise specifically stated. For example, “from 0.2 to 0.5” may mean 0.2, 0.3, 0.4, and 0.5; ranges therebetween such as 0.2-0.3, 0.3-0.4, 0.2-0.4; increments there between such as 0.25, 0.35, 0.225, 0.335, 0.49; increment ranges there between such as 0.26-0.39; and the like. The term “about” or “approximately” may refer the ordinary meaning of the term but may also indicate a value or values within about any of 1-10 percent of the listed value.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described in any way. All literature and similar materials cited in this application including, but not limited to, patents, patent applications, articles, books, treatises, and internet web pages, regardless of the format of such literature and similar materials, are expressly incorporated by reference in their entirety for any purpose. In the event that one or more of the incorporated literature and similar materials defines or uses a term in such a way that it contradicts that term's definition in this application, this application controls. While the present teachings are described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art. Certain embodiments are further described in the following examples. These embodiments are provided as examples only and are not intended to limit the scope of the claims in any way.

Aspects of this disclosure may be further understood in light of the following examples, which should not be construed as limiting the scope of the disclosure in any way.

EXAMPLES Example 1

An exemplary control reagent was prepared and tested as described below:

a) amplicons were designed comprising the fragments shown in Tables 1-3;

b) genomic sequences were selected to encompass each amplicon (the selected genomic sequences being the chromosome and nucleotide positions of the reference genome corresponding to the 5′ nucleotide of the forward and reverse primers for each amplicon and all the sequence between these two nucleotides);

c) a cassette was designed comprising an ˜400 bp EGFR sequence comprising the amplicon surrounded by (e.g., 5′ and 3′) the genomic sequence identified in step b) (the reference sequence is added in roughly equally amounts to each end of the region defined in step b) to comprise a ˜400 bp region);

d) restriction enzyme and other sites were designed to each cassette prepared in step c) (e.g., where one version may additionally include sequences that create a hairpin when the DNA is single-stranded; the restriction enzymes being chosen such that the sequences of interest are not digested but simply released from the control reagent) as shown below:

EGFR V1**

EGFR_1-ClaI-EGFR_2-HindIII-EGFR_3-SmaI-EGFR_4-XhoI-EGFR_5-NotI-EGFR_6/7-EGFR_8 **EGFR_1, etc. represt EGFR variants; restriction enzyme sites for ClaI, HindIII, SmaI, XhoI and Not I enzymes were positioned between variants.

EGFR V2***

EGFR_4-HP(7)-ClaI-EGFR_5-HP(7)-HindIII-EGFR_6/7-HP(9)-SmaI-EGFR_8 ***Hairpin 7 (HP(7)): GGGGGGGTTTTCCCCCCC (SEQ ID NO: 11); HindIII=HindIII RE site;

Hairpin 9 (HP(9)): GGGGGGGGGAACCCCCCCCC (SEQ ID NO: 12); SmaI=SmaI RE site

e) the cassette of step d) was incorporated into a common vector (pUC57) (e.g., plasmid V1) by automated synthesis of oligonucleotides on solid-phase synthesizers followed by ligation of overlapping oligonucleotides;

f) a second plasmid (e.g., “plasmid V2”) comprising multiple fragments of the gene of interest (and/or variants thereof) with a hairpin structure and a restriction site between each region (e.g., as in exemplary construct EGFR V2 above and Table 4) was also prepared by automated synthesis of oligonucleotides on solid-phase synthesizers followed by ligation of overlapping oligonucleotides;

g) the variant sequences (Tables 4-6) contained within plasmids V1 and/or V2 were then linearized HindIII;

h) the variants were then mixed with genomic DNA (e.g., wild-type gDNA) at a particular expected variant frequency (e.g., approximately 50%) (plasmid DNA and human embryonic kidney (HEK-293) genomic DNA were quantified using a fluorometer (QUBIT®) to determine the concentration; plasmid and genomic DNA were then mixed together to obtain a 1:1 molecular ratio (50% variant frequency));

i) the “variant sequences” were then tested alone to provide an expected variant frequency of 100%) to confirm sequencing; and,

j) variants of step h) were detected by NGS using the Ion Personal Genome Machine (PGM) and Illumina MiSeq (results are presented in Table 7).

Example 2 FFPE-Embedded Controls

The results of monitoring assays using FFPE-embedded controls are presented in FIGS. 4-7. As shown therein, FFPE-embedded control reagents may be used to monitor variant detection, including low frequency variants (e.g., RB1 as indicated by “C” in the figures). Variants may be tracked by the amplicon per se, GC content, sequence context, and/or variant type as desired by those of ordinary skill in the art.

Each embodiment disclosed herein may be used or otherwise combined with any of the other embodiments disclosed. Any element of any embodiment may be used in any embodiment. Although the invention has been described with reference to specific embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the true spirit and scope of the invention. In addition, modification may be made without departing from the essential teachings of the invention.

Example 3 555-Variant Control Performance Across Multiple Test Sites

A control sample was constructed that contained 555 variants from 53 different genes and tested with the Ion AMPLISEQ® Cancer Hotspot Panel v2 (CHPv2), TRUSEQ® Amplicon Cancer Panel (TSACP) and the TRUSIGHT® Tumor Panel. For each panel, two lots of the AcroMetrix® Oncology Hotspot Control were tested in duplicate, in at least two sites. Additional sites only tested one of the lots at least twice or both lots once. Sources of variation between sites may include different instruments, operators and general workflows. Also, variation in bioinformatics pipelines may have contributed significantly to variation in performance results.

FIG. 8 shows performance across different sites and panels. The average number of variants of different types detected in the ACROMETRIX® Oncology Hotspot Control are reported by site and grouped by panel. Note: The total number of variants of each type is different for each panel. See FIGS. 8-11.

To assess the detection of specific variants across different panels, twenty-two clinically-relevant variants that were targeted by three panels were selected. FIG. 9 shows detection of 22 selected variants across panels. Analysis was conducted with data from sites that tested two lots of the control at least once or one lot at least twice. Detection is indicated in dark squares and absence indicated in light squares. Site-to-site differences are apparent, even amongst those utilizing the same library preparation method, indicating the likelihood of the bioinformatics pipeline having an impact on performance.

Example 4

Performance of the control material comprising 555 variants is shown in Table 9, wherein SNV (single nucleotide variant), MNV (multiple nucleotide variant), DEL (deletion), INS (insertion), for CHPv2 (AMPLISEQ® Cancer Hotspot Panel v2), TSACP (TRUSEQ® Amplicon cancer panel), and TSTP (TRUSIGHT® tumor panel) are shown. A variant was considered to be covered by the test method if the variant was positioned between the upstream and downstream primers. A variant was considered detected if it was detected in at least one run of the control. Sanger sequencing was performed on the synthetic DNA prior to dilution with genomic DNA. Variants detected in the genomic DNA were confirmed using publicly available whole genome sequencing information for GM24385.

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

TABLE 1A Exemplary Hotspot (HS) Variants Gene name Mutation ID Mutation CDS Mutation Description Chr Start End MPL 27286 c.1514G > A Substitution - Missense 1 43814979 43814979 MPL 18918 c.1544G > T Substitution - Missense 1 43815009 43815009 MPL 27290 c.1555G > A Substitution - Missense 1 43815020 43815020 NRAS 584 c.182A > G Substitution - Missense 1 115256529 115256529 NRAS 1332933 c.174A > G Substitution - coding silent 1 115256537 115256537 NRAS 577 c.52G > A Substitution - Missense 1 115258730 115258730 NRAS 564 c.35G > A Substitution - Missense 1 115258747 115258747 NRAS 24850 c.29G > A Substitution - Missense 1 115258753 115258753 ALK 28056 c.3824G > A Substitution - Missense 2 29432664 29432664 ALK 28055 c.3522C > A Substitution - Missense 2 29443695 29443695 MSH6 13399 c.3246G > T Substitution - coding silent 2 48030632 48030632 MSH6 13395 c.3261delC Deletion - Frameshift 2 48030647 48030647 MSH6 1021299 c.3300G > A Substitution - coding silent 2 48030686 48030686 IDH1 28746 c.395G > A Substitution - Missense 2 209113112 209113112 IDH1 1404902 c.388A > G Substitution - Missense 2 209113119 209113119 IDH1 96922 c.367G > A Substitution - Missense 2 209113140 209113140 ERBB4 48362 c.2791G > T Substitution - Missense 2 212288954 212288955 ERBB4 169572 c.2782G > T Substitution - Nonsense 2 212288963 212288964 ERBB4 232263 c.1835G > A Substitution - Missense 2 212530083 212530084 ERBB4 573362 c.1828C > A Substitution - Missense 2 212530090 212530091 ERBB4 1405173 c.1784A > G Substitution - Missense 2 212530134 212530135 ERBB4 1614287 c.1089T > C Substitution - coding silent 2 212576809 212576810 ERBB4 110095 c.1022C > T Substitution - Missense 2 212576876 212576877 ERBB4 573356 c.1003G > T Substitution - Missense 2 212576895 212576896 ERBB4 1405181 c.909T > C Substitution - coding silent 2 212578347 212578348 ERBB4 160825 c.885T > G Substitution - Missense 2 212578371 212578372 ERBB4 1015994 c.829C > A Substitution - Missense 2 212587171 212587172 ERBB4 1251447 c.804C > A Substitution - Nonsense 2 212587196 212587197 ERBB4 1405184 c.730A > G Substitution - Missense 2 212589811 212589812 ERBB4 573353 c.704C > T Substitution - Missense 2 212589837 212589838 ERBB4 1015997 c.633G > A Substitution - coding silent 2 212589908 212589909 ERBB4 48369 c.542A > G Substitution - Missense 2 212652763 212652764 ERBB4 442267 c.515C > G Substitution - Missense 2 212652790 212652791 VHL 14305 c.266T > A Substitution - Missense 3 10183797 10183797 VHL 18080 c.277G > C Substitution - Missense 3 10183808 10183808 VHL 17658 c.286C > T Substitution - Nonsense 3 10183817 10183817 VHL 17886 c.296delC Deletion - Frameshift 3 10183827 10183827 VHL 17752 c.343C > A Substitution - Missense 3 10188200 10188200 VHL 14312 c.353T > C Substitution - Missense 3 10188210 10188210 VHL 14407 c.388G > C Substitution - Missense 3 10188245 10188245 VHL 14412 c.431delG Deletion - Frameshift 3 10188288 10188288 VHL 17657 c.472C > G Substitution - Missense 3 10191479 10191479 VHL 17612 c.481C > T Substitution - Nonsense 3 10191488 10191488 VHL 14311 c.499C > T Substitution - Missense 3 10191506 10191506 VHL 17837 c.506T > C Substitution - Missense 3 10191513 10191513 MLH1 26085 c.1151T > A Substitution - Missense 3 37067240 37067240 CTNNB1 5677 c.98C > G Substitution - Missense 3 41266101 41266101 CTNNB1 5662 c.110C > T Substitution - Missense 3 41266113 41266113 CTNNB1 5664 c.121A > G Substitution - Missense 3 41266124 41266124 CTNNB1 5667 c.134C > T Substitution - Missense 3 41266137 41266137 FOXL2 33661 c.402C > G Substitution - Missense 3 138665163 138665163 PIK3CA 27495 c.35G > A Substitution - Missense 3 178916648 178916648 PIK3CA 27376 c.93A > G Substitution - Missense 3 178916706 178916706 PIK3CA 1420738 c.180A > G Substitution - coding silent 3 178916793 178916793 PIK3CA 1041454 c.210C > T Substitution - coding silent 3 178916823 178916823 PIK3CA 27497 c.323G > A Substitution - Missense 3 178916936 178916936 PIK3CA 13570 c.331A > G Substitution - Missense 3 178916944 178916944 PIK3CA 125368 c.344G > T Substitution - Missense 3 178916957 178916957 PIK3CA 1420774 c.536A > G Substitution - Missense 3 178917661 178917661 PIK3CA 21462 c.971C > T Substitution - Missense 3 178921489 178921489 PIK3CA 353193 c.1002C > T Substitution - coding silent 3 178921520 178921520 PIK3CA 754 c.1035T > A Substitution - Missense 3 178921553 178921553 PIK3CA 1420804 c.1213T > C Substitution - Missense 3 178927450 178927450 PIK3CA 757 c.1258T > C Substitution - Missense 3 178927980 178927980 PIK3CA 1420828 c.1370A > G Substitution - Missense 3 178928092 178928092 PIK3CA 759 c.1616C > G Substitution - Missense 3 178936074 178936074 PIK3CA 760 c.1624G > A Substitution - Missense 3 178936082 178936082 PIK3CA 763 c.1633G > A Substitution - Missense 3 178936091 178936091 PIK3CA 1420865 c.1640A > G Substitution - Missense 3 178936098 178936098 PIK3CA 778 c.2102A > C Substitution - Missense 3 178938860 178938860 PIK3CA 769 c.2702G > T Substitution - Missense 3 178947827 178947827 PIK3CA 770 c.2725T > C Substitution - Missense 3 178947850 178947850 PIK3CA 328026 c.3110A > G Substitution - Missense 3 178952055 178952055 PIK3CA 775 c.3140A > G Substitution - Missense 3 178952085 178952085 PIK3CA 12464 c.3204_3205insA Insertion - Frameshift 3 178952149 178952150 FGFR3 715 c.746C > G Substitution - Missense 4 1803568 1803568 FGFR3 29446 c.753C > T Substitution - coding silent 4 1803575 1803575 FGFR3 723 c.850delC Deletion - Frameshift 4 1803672 1803672 FGFR3 716 c.1108G > T Substitution - Missense 4 1806089 1806089 FGFR3 24842 c.1138G > A Substitution - Missense 4 1806119 1806119 FGFR3 724 c.1150T > C Substitution - Missense 4 1806131 1806131 FGFR3 721 c.1172C > A Substitution - Missense 4 1806153 1806153 FGFR3 1428724 c.1928A > G Substitution - Missense 4 1807869 1807869 FGFR3 719 c.1948A > G Substitution - Missense 4 1807889 1807889 FGFR3 24802 c.2089G > T Substitution - Missense 4 1808331 1808331 PDGFRA 12418 c.1698_1712del15 Complex - deletion inframe 4 55141052 55141066 PDGFRA 1430085 c.1743T > C Substitution - coding silent 4 55141097 55141097 PDGFRA 22415 c.1977C > A Substitution - Missense 4 55144148 55144148 PDGFRA 1430086 c.2001A > G Substitution - coding silent 4 55144172 55144172 PDGFRA 743 c.2021C > T Substitution - Missense 4 55144547 55144547 PDGFRA 587613 c.2517G > T Substitution - coding silent 4 55152085 55152085 PDGFRA 736 c.2525A > T Substitution - Missense 4 55152093 55152093 PDGFRA 28052 c.2544C > A Substitution - Missense 4 55152112 55152112 KIT 77973 c.92C > T Substitution - Missense 4 55561702 55561702 KIT 1146 c.154G > A Substitution - Missense 4 55561764 55561764 KIT 1430106 c.218A > G Substitution - Missense 4 55561828 55561828 KIT 24637 c.1405T > C Substitution - Missense 4 55592081 55592081 KIT 41602 c.1416A > G Substitution - coding silent 4 55592092 55592092 KIT 1326 c.1509_1510insG Insertion - In frame 4 55592185 55592186 CCTAT KIT 96867 c.1516T > C Substitution - Missense 4 55592192 55592192 KIT 96885 c.1526A > T Substitution - Missense 4 55592202 55592202 KIT 1430136 c.1535A > G Substitution - Missense 4 55592211 55592211 KIT 1155 c.1588G > A Substitution - Missense 4 55593431 55593431 KIT 1275 c.1698C > T Substitution - coding silent 4 55593632 55593632 KIT 1290 c.1727T > C Substitution - Missense 4 55593661 55593661 KIT 1299 c.1755C > T Substitution - coding silent 4 55593689 55593689 KIT 1304 c.1924A > G Substitution - Missense 4 55594221 55594221 KIT 12706 c.1961T > C Substitution - Missense 4 55594258 55594258 KIT 36053 c.2089C > T Substitution - Missense 4 55595599 55595599 KIT 1430171 c.2148T > C Substitution - coding silent 4 55597500 55597500 KIT 21303 c.2209G > A Substitution - Missense 4 55597561 55597561 KIT 20402 c.2410C > T Substitution - Missense 4 55599284 55599284 KIT 19194 c.2484 + 43T > A Unknown 4 55599401 55599401 KIT 133767 c.2558G > A Substitution - Nonsense 4 55602737 55602737 KDR 35855 c.4008C > T Substitution - coding silent 4 55946171 55946171 KDR 1430203 c.3433G > A Substitution - Missense 4 55955112 55955112 KDR 48464 c.2917G > T Substitution - Missense 4 55961023 55961023 KDR 1430212 c.2619A > G Substitution - coding silent 4 55962505 55962505 KDR 32339 c.824G > T Substitution - Missense 4 55979623 55979623 FBXW7 1427592 c.2079A > G Substitution - coding silent 4 153244078 153244078 FBXW7 27083 c.2065C > T Substitution - Missense 4 153244092 153244092 FBXW7 732399 c.2033C > G Substitution - Nonsense 4 153244124 153244124 FBXW7 34018 c.2001delG Deletion - Frameshift 4 153244156 153244156 FBXW7 27913 c.1580A > G Substitution - Missense 4 153247222 153247222 FBXW7 30599 c.1576T > C Substitution - Missense 4 153247226 153247226 FBXW7 30598 c.1558G > A Substitution - Missense 4 153247244 153247244 FBXW7 34016 c.1451G > T Substitution - Missense 4 153247351 153247351 FBXW7 22974 c.1436G > A Substitution - Missense 4 153247366 153247366 FBXW7 22965 c.1394G > A Substitution - Missense 4 153249384 153249384 FBXW7 22986 c.1338G > A Substitution - Nonsense 4 153249440 153249440 FBXW7 161024 c.1322G > T Substitution - Missense 4 153249456 153249456 FBXW7 22973 c.1177C > T Substitution - Nonsense 4 153250883 153250883 FBXW7 22971 c.832C > T Substitution - Nonsense 4 153258983 153258983 FBXW7 1052125 c.744G > T Substitution - Missense 4 153259071 153259071 APC 18979 c.2543_2544insA Insertion - Frameshift 5 112173834 112173835 APC 18852 c.2626C > T Substitution - Nonsense 5 112173917 112173917 APC 19230 c.2639T > C Substitution - Missense 5 112173930 112173930 APC 19330 c.2656C > T Substitution - Nonsense 5 112173947 112173947 APC 19065 c.2752G > T Substitution - Nonsense 5 112174043 112174043 APC 13872 c.3286C > T Substitution - Nonsense 5 112174577 112174577 APC 1432250 c.3305A > G Substitution - Missense 5 112174596 112174596 APC 1432260 c.3435A > G Substitution - coding silent 5 112174726 112174726 APC 41617 c.3700delA Deletion - Frameshift 5 112174991 112174991 APC 1432280 c.3795A > G Substitution - coding silent 5 112175086 112175086 APC 19072 c.3871C > T Substitution - Nonsense 5 112175162 112175162 APC 18960 c.3880C > T Substitution - Nonsense 5 112175171 112175171 APC 18719 c.3923_3924insA Insertion - Frameshift 5 112175214 112175215 APC 18702 c.3964G > T Substitution - Nonsense 5 112175255 112175255 APC 19048 c.4057G > T Substitution - Nonsense 5 112175348 112175348 APC 19652 c.4063T > C Substitution - Missense 5 112175354 112175354 APC 18862 c.4132C > T Substitution - Nonsense 5 112175423 112175423 APC 143913 c.4141C > T Substitution - Missense 5 112175432 112175432 APC 18993 c.4189_4190delGA Deletion - Frameshift 5 112175480 112175481 APC 19087 c.4216C > T Substitution - Nonsense 5 112175507 112175507 APC 18836 c.4285C > T Substitution - Nonsense 5 112175576 112175576 APC 13864 c.4393_4394delAG Deletion - Frameshift 5 112175684 112175685 APC 1173082 c.4540delC Deletion - Frameshift 5 112175831 112175831 APC 1183180 c.4561G > T Substitution - Nonsense 5 112175852 112175852 APC 13879 c.4639G > T Substitution - Nonsense 5 112175930 112175930 APC 41616 c.4654G > T Substitution - Nonsense 5 112175945 112175945 APC 18561 c.4666_4667insA Insertion - Frameshift 5 112175957 112175958 APC 18875 c.4773_4774insA Insertion - Frameshift 5 112176064 112176065 APC 42906 c.4826C > T Substitution - Missense 5 112176117 112176117 CSF1R 947 c.2906A > G Substitution - Missense 5 149433644 149433645 CSF1R 310349 c.2878G > A Substitution - Missense 5 149433672 149433673 NPM1 17559 c.863_864insTCTG Insertion - Frameshift 5 170837547 170837548 EGFR 21683 c.323G > A Substitution - Missense 7 55211080 55211080 EGFR 174732 c.340G > A Substitution - Missense 7 55211097 55211097 EGFR 1451540 c.408C > T Substitution - coding silent 7 55211165 55211165 EGFR 21687 c.866C > T Substitution - Missense 7 55221822 55221822 EGFR 43067 c.874G > T Substitution - Missense 7 55221830 55221830 EGFR 21690 c.1793G > T Substitution - Missense 7 55233043 55233043 EGFR 35825 c.1859G > A Substitution - Missense 7 55233109 55233109 EGFR 13177 c.2063T > C Substitution - Missense 7 55241615 55241615 EGFR 41905 c.2092G > A Substitution - Missense 7 55241644 55241644 EGFR 6239 c.2156G > C Substitution - Missense 7 55241708 55241708 EGFR 13979 c.2170G > A Substitution - Missense 7 55241722 55241722 EGFR 53194 c.2197C > T Substitution - Missense 7 55242427 55242427 EGFR 13182 c.2203G > A Substitution - Missense 7 55242433 55242433 EGFR 17570 c.2222C > T Substitution - Missense 7 55242452 55242452 EGFR 6223 c.2235_2249del15 Deletion - In frame 7 55242465 55242479 EGFR 28603 c.2293G > A Substitution - Missense 7 55248995 55248995 EGFR 13190 c.2375T > C Substitution - Missense 7 55249077 55249077 EGFR 12986 c.2429G > A Substitution - Missense 7 55249131 55249131 EGFR 28610 c.2441T > C Substitution - Missense 7 55249143 55249143 EGFR 53291 c.2485G > A Substitution - Missense 7 55259427 55259427 EGFR 13424 c.2497T > G Substitution - Missense 7 55259439 55259439 EGFR 6227 c.2504A > T Substitution - Missense 7 55259446 55259446 EGFR 13430 c.2515G > A Substitution - Missense 7 55259457 55259457 EGFR 6224 c.2573T > G Substitution - Missense 7 55259515 55259515 EGFR 6213 c.2582T > A Substitution - Missense 7 55259524 55259524 EGFR 14070 c.2588G > A Substitution - Missense 7 55259530 55259530 EGFR 13008 c.2612C > G Substitution - Missense 7 55259554 55259554 MET 706 c.504G > T Substitution - Missense 7 116339642 116339642 MET 710 c.1124A > G Substitution - Missense 7 116340262 116340262 MET 29633 c.3082 + 1G > A Unknown 7 116412044 116412044 MET 1447462 c.3336T > C Substitution - coding silent 7 116417465 116417465 MET 697 c.3370C > G Substitution - Missense 7 116417499 116417499 MET 43064 c.3534G > C Substitution - Missense 7 116418969 116418969 MET 1214928 c.3562C > T Substitution - Nonsense 7 116418997 116418997 MET 1447471 c.3573T > C Substitution - coding silent 7 116419008 116419008 MET 1330154 c.3668T > G Substitution - Missense 7 116422133 116422133 MET 700 c.3757T > G Substitution - Missense 7 116423428 116423428 MET 48565 c.3778G > T Substitution - Missense 7 116423449 116423449 MET 695 c.3785A > G Substitution - Missense 7 116423456 116423456 MET 691 c.3803T > C Substitution - Missense 7 116423474 116423474 SMO 13145 c.595C > T Substitution - Missense 7 128845101 128845101 SMO 13147 c.970G > A Substitution - Missense 7 128846040 128846040 SMO 216037 c.1234C > T Substitution - Missense 7 128846398 128846398 SMO 13146 c.1604G > T Substitution - Missense 7 128850341 128850341 SMO 13150 c.1918A > G Substitution - Missense 7 128851593 128851593 BRAF 476 c.1799T > A Substitution - Missense 7 140453136 140453136 BRAF 471 c.1790T > G Substitution - Missense 7 140453145 140453145 BRAF 467 c.1781A > G Substitution - Missense 7 140453154 140453154 BRAF 462 c.1742A > G Substitution - Missense 7 140453193 140453193 BRAF 450 c.1391G > T Substitution - Missense 7 140481417 140481417 BRAF 27986 c.1380A > G Substitution - coding silent 7 140481428 140481428 BRAF 1448625 c.1359T > C Substitution - coding silent 7 140481449 140481449 BRAF 6262 c.1330C > T Substitution - Missense 7 140481478 140481478 EZH2 37028 c.1937A > T Substitution - Missense 7 148508727 148508727 FGFR1 1292693 c.816C > T Substitution - coding silent 8 38282147 38282147 FGFR1 187237 c.448C > T Substitution - Missense 8 38285864 38285864 FGFR1 1456955 c.421A > G Substitution - Missense 8 38285891 38285891 FGFR1 601 c.374C > T Substitution - Missense 8 38285938 38285938 JAK2 12600 c.1849G > T Substitution - Missense 9 5073770 5073770 JAK2 27063 c.1860C > A Substitution - Missense 9 5073781 5073781 CDKN2A 12479 c.358G > T Substitution - Nonsense 9 21971000 21971000 CDKN2A 12476 c.341C > T Substitution - Missense 9 21971017 21971017 CDKN2A 12547 c.330G > A Substitution - Nonsense 9 21971028 21971028 CDKN2A 13489 c.322G > T Substitution - Missense 9 21971036 21971036 CDKN2A 12504 c.247C > T Substitution - Missense 9 21971111 21971111 CDKN2A 12475 c.238C > T Substitution - Nonsense 9 21971120 21971120 CDKN2A 13281 c.205G > T Substitution - Nonsense 9 21971153 21971153 CDKN2A 12473 c.172C > T Substitution - Nonsense 9 21971186 21971186 GNAQ 1110323 c.1002C > T Substitution - coding silent 9 80336317 80336317 GNAQ 52975 c.548G > A Substitution - Missense 9 80412493 80412493 GNAQ 1463119 c.523A > T Substitution - Missense 9 80412518 80412518 ABL1 12631 c.742C > G Substitution - Missense 9 133738342 133738342 ABL1 12577 c.749G > A Substitution - Missense 9 133738349 133738349 ABL1 12576 c.757T > C Substitution - Missense 9 133738357 133738357 ABL1 12573 c.763G > A Substitution - Missense 9 133738363 133738363 ABL1 12602 c.827A > G Substitution - Missense 9 133747520 133747520 ABL1 235737 c.878_879insGCC Complex - insertion inframe 9 133747571 133747572 ABL1 12578 c.1052T > C Substitution - Missense 9 133748391 133748391 ABL1 12611 c.1064A > G Substitution - Missense 9 133748403 133748403 ABL1 12605 c.1075T > G Substitution - Missense 9 133748414 133748414 ABL1 49071 c.1150C > A Substitution - Missense 9 133750319 133750319 ABL1 12604 c.1187A > G Substitution - Missense 9 133750356 133750356 NOTCH1 87862 c.7412C > A Substitution - Nonsense 9 139390779 139390779 NOTCH1 13070 c.7386delC Deletion - Frameshift 9 139390805 139390805 NOTCH1 12776 c.7375C > T Substitution - Nonsense 9 139390816 139390816 NOTCH1 13061 c.7318C > T Substitution - Nonsense 9 139390873 139390873 NOTCH1 13048 c.5033T > C Substitution - Missense 9 139397768 139397768 NOTCH1 308587 c.5025C > T Substitution - coding silent 9 139397776 139397776 NOTCH1 12771 c.4799T > C Substitution - Missense 9 139399344 139399344 NOTCH1 13053 c.4793G > C Substitution - Missense 9 139399350 139399350 NOTCH1 13042 c.4778T > C Substitution - Missense 9 139399365 139399365 NOTCH1 12772 c.4721T > C Substitution - Missense 9 139399422 139399422 RET 29803 c.1852T > C Substitution - Missense 10 43609096 43609096 RET 29804 c.1858T > C Substitution - Missense 10 43609102 43609102 RET 1048 c.1894_1906 > AGCT Complex - deletion inframe 10 43609942 43609954 RET 1223553 c.1942G > A Substitution - Missense 10 43609990 43609990 RET 976 c.1991C > A Substitution - Missense 10 43610039 43610039 RET 21338 c.2304G > C Substitution - Missense 10 43613840 43613840 RET 977 c.2647_2648GC > TT Substitution - Missense 10 43615568 43615569 RET 963 c.2701G > A Substitution - Missense 10 43615622 43615622 RET 965 c.2753T > C Substitution - Missense 10 43617416 43617416 PTEN 5298 c.19G > T Substitution - Nonsense 10 89624245 89624245 PTEN 5101 c.40A > G Substitution - Missense 10 89624266 89624266 PTEN 5153 c.49C > T Substitution - Nonsense 10 89624275 89624275 PTEN 5107 c.71A > G Substitution - Missense 10 89624297 89624297 PTEN 5134 c.80A > G Substitution - Missense 10 89653782 89653782 PTEN 5142 c.112C > T Substitution - Missense 10 89653814 89653814 PTEN 5050 c.142A > G Substitution - Missense 10 89653844 89653844 PTEN 1349479 c.156T > C Substitution - coding silent 10 89653858 89653858 PTEN 5129 c.163A > G Substitution - Missense 10 89653865 89653865 PTEN 5257 c.166T > G Substitution - Missense 10 89685271 89685271 PTEN 5036 c.202T > C Substitution - Missense 10 89685307 89685307 PTEN 5916 c.209 + 5G > A Unknown 10 89685319 89685319 PTEN 5102 c.212G > A Substitution - Missense 10 89690805 89690805 PTEN 4956 c.227_228delAT Deletion - Frameshift 10 89690820 89690821 PTEN 5205 c.245A > C Substitution - Missense 10 89690838 89690838 PTEN 5983 c.253 + 1G > A Unknown 10 89690847 89690847 PTEN 5139 c.263A > G Substitution - Missense 10 89692779 89692779 PTEN 5109 c.302T > C Substitution - Missense 10 89692818 89692818 PTEN 5266 c.314G > T Substitution - Missense 10 89692830 89692830 PTEN 5199 c.334C > G Substitution - Missense 10 89692850 89692850 PTEN 5123 c.395G > A Substitution - Missense 10 89692911 89692911 PTEN 5130 c.449A > G Substitution - Missense 10 89692965 89692965 PTEN 5144 c.464A > G Substitution - Missense 10 89692980 89692980 PTEN 5287 c.477G > T Substitution - Missense 10 89692993 89692993 PTEN 5907 c.493-12delT Unknown 10 89711863 89711863 PTEN 35406 c.578T > C Substitution - Missense 10 89711960 89711960 PTEN 4978 c.595_597delATG Deletion - In frame 10 89711977 89711979 PTEN 5279 c.610C > A Substitution - Missense 10 89711992 89711992 PTEN 5072 c.615G > A Substitution - Missense 10 89711997 89711997 PTEN 5154 c.697C > T Substitution - Nonsense 10 89717672 89717672 PTEN 5292 c.703G > T Substitution - Nonsense 10 89717678 89717678 PTEN 35849 c.721T > C Substitution - Missense 10 89717696 89717696 PTEN 43075 c.787A > T Substitution - Nonsense 10 89717762 89717762 PTEN 5809 c.800delA Deletion - Frameshift 10 89717775 89717775 PTEN 1349606 c.879A > G Substitution - coding silent 10 89720728 89720728 PTEN 5312 c.895G > T Substitution - Nonsense 10 89720744 89720744 PTEN 4958 c.955_958delACTT Deletion - Frameshift 10 89720804 89720807 PTEN 1349625 c.1025A > G Substitution - Missense 10 89720874 89720874

  

 

 

PTEN 4936 c.1040_1041delTC Deletion - Frameshift 10 89725057 89725058 PTEN 1349633 c.1055A > G Substitution - Missense 10 89725072 89725072 PTEN 23645 c.1091C > G Substitution - Missense 10 89725108 89725108 FGFR2 36912 c.1647T > A Substitution - Missense 10 123258034 123258034 FGFR2 36906 c.1144T > C Substitution - Missense 10 123274774 123274774 FGFR2 36904 c.1124A > G Substitution - Missense 10 123274794 123274794 FGFR2 1346272 c.1108A > G Substitution - Missense 10 123274810 123274810 FGFR2 36901 c.929A > G Substitution - Missense 10 123279503 123279503 FGFR2 29824 c.913G > A Substitution - Missense 10 123279519 123279519 FGFR2 36903 c.755C > G Substitution - Missense 10 123279677 123279677 HRAS 499 c.182A > G Substitution - Missense 11 533874 533874 HRAS 495 c.175G > A Substitution - Missense 11 533881 533881 HRAS 249860 c.81T > C Substitution - coding silent 11 534242 534242 HRAS 483 c.35G > T Substitution - Missense 11 534288 534288 ATM 21323 c.1009C > T Substitution - Missense 11 108117798 108117798 ATM 21825 c.1229T > C Substitution - Missense 11 108119823 108119823 ATM 22499 c.1810C > T Substitution - Missense 11 108123551 108123551 ATM 1158828 c.1898 + 2T > A Unknown 11 108123641 108123641 ATM 21826 c.2572T > C Substitution - Missense 11 108138003 108138003 ATM 22507 c.3925G > A Substitution - Missense 11 108155132 108155132 ATM 21920 c.5044G > T Substitution - Missense 11 108170479 108170479 ATM 218294 c.5152C > G Substitution - Missense 11 108170587 108170587 ATM 49005 c.5178-1G > T Unknown 11 108172374 108172374 ATM 172204 c.5188C > T Substitution - Nonsense 11 108172385 108172385 ATM 21918 c.5224G > C Substitution - Missense 11 108172421 108172421 ATM 12792 c.5380C > T Substitution - coding silent 11 108173640 108173640 ATM 1183962 c.5476T > G Substitution - Missense 11 108173736 108173736 ATM 21922 c.5821G > C Substitution - Missense 11 108180945 108180945 ATM 12951 c.7325A > C Substitution - Missense 11 108200958 108200958 ATM 12791 c.7996A > G Substitution - Missense 11 108204681 108204681 ATM 21636 c.8084G > C Substitution - Missense 11 108205769 108205769 ATM 1235404 c.8095C > A Substitution - Missense 11 108205780 108205780 ATM 22481 c.8174A > T Substitution - Missense 11 108206594 108206594 ATM 1183939 c.8624A > G Substitution - Missense 11 108218045 108218045 ATM 22485 c.8668C > G Substitution - Missense 11 108218089 108218089 ATM 21930 c.8839A > T Substitution - Missense 11 108225590 108225590 ATM 21626 c.9023G > A Substitution - Missense 11 108236087 108236087 ATM 1351060 c.9054A > G Substitution - coding silent 11 108236118 108236118 ATM 21624 c.9139C > T Substitution - Nonsense 11 108236203 108236203 KRAS 41307 c.491G > A Substitution - Missense 12 25362805 25362805 KRAS 19940 c.351A > C Substitution - Missense 12 25378647 25378647 KRAS 554 c.183A > C Substitution - Missense 12 25380275 25380275 KRAS 546 c.175G > A Substitution - Missense 12 25380283 25380283 KRAS 1169214 c.101C > T Substitution - Missense 12 25398207 25398207 KRAS 14208 c.104C > T Substitution - Missense 12 25398215 25398215 KRAS 521 c.35G > A Substitution - Missense 12 25398284 25398284 KRAS 507 c.24A > G Substitution - coding silent 12 25398295 25398295 PTPN11 13011 c.181G > T Substitution - Missense 12 112888165 112888165 PTPN11 13013 c.205G > A Substitution - Missense 12 112888189 112888189 PTPN11 13015 c.215C > T Substitution - Missense 12 112888199 112888199 PTPN11 13000 c.226G > A Substitution - Missense 12 112888210 112888210 PTPN11 13034 c.1472C > T Substitution - Missense 12 112926852 112926852 PTPN11 13027 c.1508G > C Substitution - Missense 12 112926888 112926888 PTPN11 1358900 c.1519A > G Substitution - Missense 12 112926899 112926899 PTPN11 13031 c.1528C > A Substitution - Missense 12 112926908 112926908 HNF1A 21471 c.617G > T Substitution - Missense 12 121431413 121431413 HNF1A 24900 c.632A > C Substitution - Missense 12 121431428 121431428 HNF1A 24832 c.685C > T Substitution - Nonsense 12 121431481 121431481 HNF1A 21474 c.710A > G Substitution - Missense 12 121431506 121431506 HNF1A 24923 c.779C > T Substitution - Missense 12 121432032 121432032 HNF1A 24692 c.787C > T Substitution - Missense 12 121432040 121432040 HNF1A 21481 c.872_873insC Insertion - Frameshift 12 121432125 121432126 FLT3 1166729 c.2516A > G Substitution - Missense 13 28592629 28592629 FLT3 783 c.2503G > T Substitution - Missense 13 28592642 28592642 FLT3 25248 c.2492G > A Substitution - Missense 13 28592653 28592653 FLT3 786 c.2039C > T Substitution - Missense 13 28602329 28602329 FLT3 27907 c.1800_1801ins21 Insertion - In frame 13 28608255 28608256 FLT3 19522 c.1775T > C Substitution - Missense 13 28608281 28608281 FLT3 28042 c.1352C > T Substitution - Missense 13 28610138 28610138 RB1 890 c.409G > T Substitution - Nonsense 13 48919244 48919244 RB1 915 c.596T > A Substitution - Nonsense 13 48923148 48923148 RB1 28816 c.940-2A > T Unknown 13 48941628 48941628 RB1 891 c.958C > T Substitution - Nonsense 13 48941648 48941648 RB1 1367204 c.968A > G Substitution - Missense 13 48941658 48941658 RB1 1367206 c.982A > G Substitution - Missense 13 48941672 48941672 RB1 879 c.1072C > T Substitution - Nonsense 13 48942685 48942685 RB1 895 c.1363C > T Substitution - Nonsense 13 48953760 48953760 RB1 887 c.1654C > T Substitution - Nonsense 13 48955538 48955538 RB1 888 c.1666C > T Substitution - Nonsense 13 48955550 48955550 RB1 1367255 c.1687T > C Substitution - Missense 13 48955571 48955571 RB1 892 c.1735C > T Substitution - Nonsense 13 49027168 49027168 RB1 35483 c.1814 + 2T > C Unknown 13 49027249 49027249 RB1 870 c.2028_2040del13 Deletion - Frameshift 13 49033891 49033903 RB1 13117 c.2053C > T Substitution - Nonsense 13 49033916 49033916 RB1 942 c.2063T > C Substitution - Missense 13 49033926 49033926 RB1 1042 c.2107-2A > G Unknown 13 49037865 49037865 RB1 883 c.2117G > T Substitution - Missense 13 49037877 49037877 RB1 940 c.2143A > T Substitution - Nonsense 13 49037903 49037903 RB1 1367309 c.2153A > G Substitution - Missense 13 49037913 49037913 RB1 868 c.2242G > T Substitution - Nonsense 13 49039164 49039164 RB1 916 c.2261T > G Substitution - Missense 13 49039183 49039183 RB1 551465 c.2267A > G Substitution - Missense 13 49039189 49039189 RB1 254910 c.2293A > T Substitution - Nonsense 13 49039215 49039215 AKT1 33765 c.49G > A Substitution - Missense 14 105246551 105246551 IDH2 33733 c.515G > A Substitution - Missense 15 90631838 90631838 IDH2 1375400 c.474A > G Substitution - coding silent 15 90631879 90631879 IDH2 41590 c.419G > A Substitution - Missense 15 90631934 90631934 CDH1 1379165 c.1058A > G Substitution - Missense 16 68846087 68846087 CDH1 19748 c.1108G > C Substitution - Missense 16 68846137 68846137 CDH1 19750 c.1204G > A Substitution - Missense 16 68847282 68847282 CDH1 25267 c.1733_1734insC Insertion - Frameshift 16 68855925 68855926 CDH1 19746 c.1742T > C Substitution - Missense 16 68855934 68855934 CDH1 19758 c.1774G > A Substitution - Missense 16 68855966 68855966 CDH1 19743 c.1849G > A Substitution - Missense 16 68856041 68856041 CDH1 19822 c.1901C > T Substitution - Missense 16 68856093 68856093 CDH1 19418 c.1913G > A Substitution - Nonsense 16 68856105 68856105 TP53 13747 c.1146delA Deletion - Frameshift 17 7572963 7572963 TP53 307348 c.1123C > T Substitution - Nonsense 17 7572986 7572986 TP53 1191161 c.1101-2A > G Unknown 17 7573010 7573010 TP53 11073 c.1024C > T Substitution - Nonsense 17 7574003 7574003

 

 

 

TP53 11071 c.1009C > T Substitution - Missense 17 7574018 7574018 TP53 11514 c.1001G > T Substitution - Missense 17 7574026 7574026 TP53 11354 c.991C > T Substitution - Nonsense 17 7576855 7576855 TP53 44823 c.981T > G Substitution - Nonsense 17 7576865 7576865 TP53 46088 c.963A > G Substitution - coding silent 17 7576883 7576883 TP53 10786 c.949C > T Substitution - Nonsense 17 7576897 7576897 TP53 10663 c.916C > T Substitution - Nonsense 17 7577022 7577022 TP53 10710 c.892G > T Substitution - Nonsense 17 7577046 7577046 TP53 10863 c.833C > T Substitution - Missense 17 7577105 7577105 TP53 10660 c.818G > A Substitution - Missense 17 7577120 7577120 TP53 10662 c.743G > A Substitution - Missense 17 7577538 7577538 TP53 6932 c.733G > A Substitution - Missense 17 7577548 7577548 TP53 10812 c.722C > T Substitution - Missense 17 7577559 7577559 TP53 10725 c.701A > G Substitution - Missense 17 7577580 7577580 TP53 10758 c.659A > G Substitution - Missense 17 7578190 7578190 TP53 44317 c.653T > A Substitution - Missense 17 7578196 7578196 TP53 10667 c.646G > A Substitution - Missense 17 7578203 7578203 TP53 43947 c.614A > G Substitution - Missense 17 7578235 7578235 TP53 10738 c.542G > A Substitution - Missense 17 7578388 7578388 TP53 10808 c.488A > G Substitution - Missense 17 7578442 7578442 TP53 10739 c.481G > A Substitution - Missense 17 7578449 7578449 TP53 10670 c.469G > T Substitution - Missense 17 7578461 7578461 TP53 10801 c.404G > A Substitution - Missense 17 7578526 7578526 TP53 11582 c.395A > G Substitution - Missense 17 7578535 7578535 TP53 11462 c.388C > G Substitution - Missense 17 7578542 7578542 TP53 44226 c.380C > T Substitution - Missense 17 7578550 7578550 TP53 44985 c.375 + 17G > A Unknown 17 7579295 7579295 TP53 43904 c.375G > A Substitution - coding silent 17 7579312 7579312 TP53 10716 c.329G > T Substitution - Missense 17 7579358 7579358 TP53 46103 c.319T > G Substitution - Missense 17 7579368 7579368 TP53 44492 c.273G > A Substitution - Nonsense 17 7579414 7579414 TP53 43910 c.245C > T Substitution - Missense 17 7579442 7579442 TP53 12168 c.166G > T Substitution - Nonsense 17 7579521 7579521 TP53 44907 c.151G > T Substitution - Nonsense 17 7579536 7579536 TP53 43664 c.134T > C Substitution - Missense 17 7579553 7579553 TP53 46286 c.112C > T Substitution - Nonsense 17 7579575 7579575 TP53 85573 c.80delC Deletion - Frameshift 17 7579716 7579716 ERBB2 14060 c.2264T > C Substitution - Missense 17 37880220 37880220 ERBB2 1251412 c.2305G > T Substitution - Missense 17 37880261 37880261 ERBB2 20959 c.2324_2325ins12 Insertion - In frame 17 37880995 37880996 ERBB2 14065 c.2524G > A Substitution - Missense 17 37881332 37881332 ERBB2 686 c.2570A > G Substitution - Missense 17 37881378 37881378 ERBB2 21985 c.2632C > T Substitution - Missense 17 37881440 37881440 SMAD4 1389031 c.306T > C Substitution - coding silent 18 48575112 48575112 SMAD4 14229 c.377T > C Substitution - Missense 18 48575183 48575183 SMAD4 218557 c.389C > T Substitution - Missense 18 48575195 48575195 SMAD4 14168 c.403C > T Substitution - Nonsense 18 48575209 48575209 SMAD4 13115 c.431C > G Substitution - Nonsense 18 48575671 48575671 SMAD4 14118 c.502G > T Substitution - Nonsense 18 48581198 48581198 SMAD4 1226725 c.533C > A Substitution - Nonsense 18 48581229 48581229 SMAD4 308153 c.547C > T Substitution - Nonsense 18 48581243 48581243 SMAD4 14057 c.733C > T Substitution - Nonsense 18 48584560 48584560 SMAD4 22901 c.766C > T Substitution - Nonsense 18 48584593 48584593 SMAD4 14217 c.776_777delCT Deletion - Frameshift 18 48584603 48584604 SMAD4 14163 c.931C > T Substitution - Nonsense 18 48586262 48586262 SMAD4 14167 c.955 + 5G > C Unknown 18 48586291 48586291 SMAD4 1389054 c.1001A > G Substitution - Missense 18 48591838 48591838 SMAD4 1389057 c.1010A > G Substitution - Missense 18 48591847 48591847 SMAD4 14109 c.1018A > G Substitution - Missense 18 48591855 48591855 SMAD4 14111 c.1028C > G Substitution - Nonsense 18 48591865 48591865 SMAD4 14249 c.1156G > C Substitution - Missense 18 48593405 48593405 SMAD4 14103 c.1216G > A Substitution - Missense 18 48593465 48593465 SMAD4 14223 c.1229_1230insCA Insertion - Frameshift 18 48593478 48593479 SMAD4 1389077 c.1248A > G Substitution - coding silent 18 48593497 48593497 SMAD4 14096 c.1333C > T Substitution - Nonsense 18 48603032 48603032 SMAD4 14114 c.1504A > G Substitution - Missense 18 48604682 48604682 SMAD4 1389099 c.1519A > G Substitution - Missense 18 48604697 48604697 SMAD4 14134 c.1576G > T Substitution - Nonsense 18 48604754 48604754 SMAD4 1389106 c.1591C > A Substitution - coding silent 18 48604769 48604769 STK11 21212 c.169delG Deletion - Frameshift 19 1207077 1207077 STK11 21570 c.465-1G > T Unknown 19 1220371 1220371 STK11 27316 c.475C > T Substitution - Nonsense 19 1220382 1220382 STK11 20944 c.580G > T Substitution - Missense 19 1220487 1220487 STK11 25229 c.595G > T Substitution - Nonsense 19 1220502 1220502 STK11 29005 c.816C > T Substitution - coding silent 19 1221293 1221293 STK11 21355 c.842C > T Substitution - Missense 19 1221319 1221319 STK11 21360 c.1062C > G Substitution - Missense 19 1223125 1223125 GNA11 21651 c.547C > T Substitution - Missense 19 3115012 3115012 GNA11 52969 c.626A > T Substitution - Missense 19 3118942 3118942 JAK3 34213 c.2164G > A Substitution - Missense 19 17945696 17945696 JAK3 34214 c.1715C > T Substitution - Missense 19 17948009 17948009 SRC 1227526 c.1460C > T Substitution - Missense 20 36031630 36031631 GNAS 244725 c.489C > T Substitution - coding silent 20 57480494 57480494 GNAS 27887 c.601C > T Substitution - Missense 20 57484420 57484420 GNAS 27888 c.680A > T Substitution - Missense 20 57484596 57484596 SMARCB1 1002 c.118C > T Substitution - Nonsense 22 24133967 24133967 SMARCB1 991 c.141C > A Substitution - Nonsense 22 24133990 24133990 SMARCB1 24595 c.157C > T Substitution - Nonsense 22 24134006 24134006 SMARCB1 992 c.472C > T Substitution - Nonsense 22 24143240 24143240 SMARCB1 51386 c.566_567ins19 Insertion - Frameshift 22 24145547 24145548 SMARCB1 993 c.601C > T Substitution - Nonsense 22 24145582 24145582 SMARCB1 999 c.607G > A Substitution - Missense 22 24145588 24145588 SMARCB1 1057 c.1148delC Deletion - Frameshift 22 24176357 24176357

TABLE 1B Exemplary Copy Number Variants (CNV) Gene Name Chromosome Start End ERBB2 chr17 37845134 37845207 ERBB2 chr17 37852282 37852381 ERBB2 chr17 37860184 37860303 ERBB2 chr17 37871503 37871582 ERBB2 chr17 37876682 37876784 ERBB2 chr17 37884464 37884584 ERBB2 chr17 37854903 37855025 ERBB2 chr17 37884065 37884183 ERBB2 chr17 37866483 37866606 ERBB2 chr17 37880963 37881086 KRAS chr12 25378600 25378682 PDGFRA chr4 55140973 55141093

TABLE 2 Control Reagent* Sequence A B C D E F G H 1 CSF1R APC APC APC APC CSF1R APC APC 2 EGFR EGFR CSF1R CSF1R CSF1R EGFR EGFR CSF1R 3 FBXW7 FBXW7 EGFR FGFR3 EGFR FGFR1 FGFR3 EGFR 4 FGFR3 FGFR3 ERBB4 FLT3 FBXW7 FGFR3 FLT3 FGFR3 5 FLT3 FLT3 FGFR3 KDR FGFR3 FLT3 HRAS FLT3 6 GNA11 KDR FLT3 KRAS FLT3 HRAS IDH1 HRAS 7 HNF1A KRAS HRAS PDGFRA HRAS KDR KDR IDH1 8 HRAS PDGFRA KDR RET KRAS KIT KRAS KRAS 9 PDGFRA PIK3CA KIT STK11 PDGFRA KRAS PDGFRA PDGFRA 10 PIK3CA RET KRAS TP53 RET MET RET PIK3CA 11 RET TP53 PDGFRA — SMAD4 NOTCH1 TP53 RET 12 STK11 — RET — TP53 PDGFRA — STK11 13 TP53 — SMAD4 — — PIK3CA — TP53 14 VHL — TP53 — — SMARCB1 — — 15 — — — — — SMO — — 16 — — — — — TP53 — — *APC (Adenomatous polyposis coli, deleted in polyposis 2.5 (DP2.5); Chr. 5: 112.04-112.18 Mb; Ref. Seq. NM_000038 and NP_000029), CSF1R (Colony stimulating factor 1 receptor, macrophage colony-stimulating factor receptor (M-CSFR), CD115; Chr. 5, 149.43-149.49 Mb; Ref. Seq. NM_005211 and NM_005202), EGFR (epidermal growth factor receptor; Chr. 7: 55.09-55.32 Mb; RefSeq Nos. NM_005228 and NP_0052219), FBXW7 (F-box/WD repeat-containing protein 7; Chr. 4: 153.24-153.46 Mb; RefSeq. Nos. NM_001013415 and NP_001013433), FGFR1 (Fibroblast growth factor receptor 1, basic fibroblast growth factor receptor 1, fms-related tyrosine kinase-2 / Pfeiffer syndrome, CD331; Chr. 8: 38.27-38.33 Mb; RefSeq. Nos. NM_001174063 and NP_001167534), FGFR3 (Fibroblast growth factor receptor 3, CD333; chr. 4: 1.8-1.81 Mb; RefSeq Nos. NM_000142 and NP_000133), FLT3 (Fms-like tyrosine kinase 3, CD135, fetal liver kinase-2 (Flk2); Chr. 13: 28.58-28.67 Mb; RefSeq Nos. NM_004119 and NP_004110), GNA11 (Guanine nucleotide-binding protein subunit alpha-11; Chr. 19: 3.09-3.12 Mb; RefSeq Nos. NM_002067 and NP_002058), HNF1A (hepatocyte nuclear factor 1 homeobox A; Chr. 12: 121.42-121.44 Mb; RefSeq Nos. NM_000545 and NP_000536), HRAS (GTPase HRas, transforming protein p21; Chr. 11: 0.53-0.54 Mb; RefSeq Nos. NM_001130442 and NP_001123914), IDH1 (Isocitrate dehydrogenase 1 (NADP+), soluble; Chr. 2: 209.1-209.13 Mb; RefSeq Nos. NM_005896 and NP_005887), KDR (Kinase insert domain receptor, vascular endothelial growth factor receptor 2, CD309; Chr. 4: 55.94-55.99 Mb; RefSeq Nos. NM_002253 and NP_002244), KIT (Mast/stem cell growth factor receptor (SCFR), proto-oncogene c-Kit, tyrosine-protein kinase Kit, CD117; Chr. 4: 55.52-55.61 Mb; RefSeq Nos. NM_000222 and NP_000213), KRAS (GTPase KRas, V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog; Chr. 12: 25.36-25.4 Mb; RefSeq Nos. NM_004985-NP_004976), MET (c-Met, MNNG HOS Transforming gene, hepatocyte growth factor receptor; Chr. 7: 116.31-116.44 Mb; RefSeq Nos. NM_000245 and NP_000236), NOTCH1 (Notch homolog 1, translocation-associated (Drosophila); Chr. 9: 139.39-139.44; RefSeq Nos. NM_017617 and NP_060087), PDGFRA (Alpha-type platelet-derived growth factor receptor; Chr. 4: 55.1-55.16 Mb; RefSeq Nos. NM_006206 and NP_006197), PIK3CA (p110α protein; Chr. 3: 178.87-178.96 Mb; RefSeq Nos. NM_006218 and NP_006209), RET (receptor tyrosine kinase; Chr. 10: 43.57-43.64; RefSeq Nos. NM_000323 and NP_065681), SMAD4 (Chr. 18: 48.49-48.61 Mb; RefSeq Nos. NM_005359 and NP_005350), SMARCB1 (SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily B member 1; Chr. 22: 24.13-24.18 Mb; RefSeq Nos. NM_001007468 and NP_001007469), SMO (Smoothened; Chr. 7: 128.83-128.85 Mb; RefSeq Nos. NM_005631 and NP_005622), STK11 (Serine/threonine kinase 11, liver kinase B1 (LKB1), renal carcinoma antigen NY-REN-19; Chr. 19: 1.19-1.23 Mb; RefSeq Nos. NM_000455 and NP_000446), TP53 (protein 53, tumor protein 53; Chr. 17: 7.57-7.59 Mb; RefSeq Nos. NM_000546 and NP_000537), VHL (Von Hippel-Lindau tumor suppressor; Chr. 3: 10.18-10.19 Mb; RefSeq Nos. NM_000551 and NP_000542).

One or more variants of each of these reference sequences may also be represented in each control sequence and/or control reagent. In some embodiments, for instance, multiple variants may be included for each reference sequence. Panels of reference sequences may also be designed to represent particular metabolic, genetic information processing, environmental information processing, cellular process, organismal system, disease, drug development, or other pathways (e.g., KEGG pathways (http://www.genome.jp/kegg/pathway.html, Nov. 8, 2013)). Control reagents such as these may be assayed separately or combined into a single assay. The control reagents may also be designed to include various amounts of each reference sequences and/or variants thereof.

TABLE 3 Number of Number of Variants Variants Run ID Detected Expected Detection Rate Bad Run  8 15  53% Bad Run 12 15  80% Good Run 15 15 100% Good Run 15 15 100% Good Run 15 15 100% Good Run 15 15 100% Good Run 15 15 100% Good Run 15 15 100%

TABLE 4 Included in Included in # of Plasmid Plasmid Fragment ID Variants V1 V2 EGFR_1 1 Yes No EGFR_2 2 Yes No EGFR_3 2 Yes No EGFR_4 13 Yes Yes EGFR_5 7 Yes Yes EGFR_6/7 10 Yes Yes EGFR_8 8 Yes Yes

TABLE 5 Included variants for each EGFR fragment EGFR_1 EGFR _2 EGFR _3 EGFR_4 EGFR_5 EGFR_6_7 EGFR _8 COSM21683 COSM21686 COSM21689 COSM41905 COSM13180 COSM28603 COSM6224 COSM21687 COSM21690 COSM28508 COSM53194 COSM26445 COSM12675 COSM28511 COSM18419 COSM6241 COSM6213 COSM12988 COSM13182 COSM12376 COSM14070 COSM13427 COSM17570 COSM12381 COSM28607 COSM41603 COSM6223 COSM13007 COSM33725 COSM28601 COSM21984 COSM6240 COSM13008 COSM6252 COSM13192 COSM26438 COSM6239 COSM28610 COSM12373 COSM41663 COSM22992 COSM28510 COSM13979

TABLE 6 Mutation Detail Mutation Gene Mutation Mutation GRCh37 ID name ID Mutation CDS Mutation AA Description Chr Start End strand 21683 EGFR 21683 c.323G > A p.R108K Substitution- 7 55211080 55211080 + Missense 21686 EGFR 21686 c.865G > A p.A289T Substitution- 7 55221821 55221821 + Missense 21687 EGFR 21687 c.866C > T p.A289V Substitution- 7 55221822 55221822 + Missense 21689 EGFR 21689 c.1787C > T p.P596L Substitution- 7 55233037 55233037 + Missense 21690 EGFR 21690 c.1793G > T p.G598V Substitution- 7 55233043 55233043 + Missense 41905 EGFR 41905 c.2092G > A p.A698T Substitution- 7 55241644 55241644 + Missense 28508 EGFR 28508 c.2104G > T p.A702S Substitution- 7 55241656 55241656 + Missense 28511 EGFR 28511 c.2108T > C p.L703P Substitution- 7 55241660 55241660 + Missense 12988 EGFR 12988 c.2125G > A p.E709K Substitution- 7 55241677 55241677 + Missense 13427 EGFR 13427 c.2126A > C p.E709A Substitution- 7 55241678 55241678 + Missense 41603 EGFR 41603 c.2134T > C p.F712L Substitution- 7 55241686 55241686 + Missense 28601 EGFR 28601 c.2135T > C p.F712S Substitution- 7 55241687 55241687 + Missense 6252 EGFR 6252 c.2155G > A p.G719S Substitution- 7 55241707 55241707 + Missense 6239 EGFR 6239 c.2156G > C p.G719A Substitution- 7 55241708 55241708 + Missense 12373 EGFR 12373 c.2159C > T p.S720F Substitution- 7 55241711 55241711 + Missense 22992 EGFR 22992 c.2161G > A p.G721S Substitution- 7 55241713 55241713 + Missense 28510 EGFR 28510 c.2162G > C p.G721A Substitution- 7 55241714 55241714 + Missense 13979 EGFR 13979 c.2170G > A p.G724S Substitution- 7 55241722 55241722 + Missense 13180 EGFR 13180 c.2188C > T p.L730F Substitution- 7 55242418 55242418 + Missense 53194 EGFR 53194 c.2197C > T p.P733S Substitution- 7 55242427 55242427 + Missense 18419 EGFR 18419 c.2200G > A p.E734K Substitution- 7 55242430 55242430 + Missense 13182 EGFR 13182 c.2203G > A p.G735S Substitution- 7 55242433 55242433 + Missense 17570 EGFR 17570 c.2222C > T p.P741L Substitution- 7 55242452 55242452 + Missense 6223 EGFR 6223 c.2235_2249dell5 p.E746_A750delELREA Deletion- 7 55242465 55242479 + In frame 21984 EGFR 21984 c.2281G > T p.D761Y Substitution- 7 55242511 55242511 + Missense 28603 EGFR 28603 c.2293G > A p.V765M Substitution- 7 55248995 55248995 + Missense 26445 EGFR 26445 c.2300C > T p.A767V Substitution- 7 55249002 55249002 + Missense 6241 EGFR 6241 c.2303G > T p.57681 Substitution- 7 55249005 55249005 + Missense 12376 EGFR 12376 c.2307_2308insGCCAGCGTG p.V769_D770insASV Insertion- 7 55249009 5249010 + In frame 12381 EGFR 12381 c.2319_2320insAACCCCCAC p.H773_V774insNPH Insertion- 7 55249021 55249022 + In frame 13007 EGFR 13007 c.2335_2336GG > TT p.G779F Substitution- 7 55249037 55249038 + Missense 6240 EGFR 6240 c.2369C > T p.T790M Substitution- 7 55249071 55249071 + Missense 13192 EGFR 13192 c.2428G > A p.G810S Substitution- 7 55249130 55249130 + Missense 28610 EGFR 28610 c.2441T > C p.L814P Substitution- 7 55249143 55249143 + Missense 41663 EGFR 41663 c.2462T > C p.I821T Substitution- 7 55249164 55249164 + Missense 6224 EGFR 6224 c.2573T > G p.L858R Substitution- 7 55259515 55259515 + Missense 12675 EGFR 12675 c.2575G > A p.A859T Substitution- 7 55259517 55259517 + Missense 6213 EGFR 6213 c.2582T > A p.L861Q Substitution- 7 55259524 55259524 + Missense 14070 EGFR 14070 c.2588G > A p.G863D Substitution- 7 55259530 55259530 + Missense 28607 EGFR 28607 c.2603A > G p.E868G Substitution- 7 55259545 55259545 + Missense 33725 EGFR 33725 c.2609A > G p.H870R Substitution- 7 55259551 55259551 + Missense 13008 EGFR 13008 c.2612C > G p.A871G Substitution- 7 55259554 55259554 + Missense 26438 EGFR 26438 c.2620G > A p.G874S Substitution- 7 55259562 55259562 + Missense

TABLE 7 Results of EGFR plasmid EGFR Plasmid V1 EGFR Plasmid V2 Mutation Ion AmpliSeq Illumina Ion AmpliSeq Illumina ID CHP2 TruSeq CHP2 TruSeq 21683 Called Called Not Included Not Included 21686 Called Called Not Included Not Included 21687 Called Called Not Included Not Included 21689 Called Not Targeted Not Included Not Included 21690 Called Called Not Included Not Included 41905 Called Not Called** Called Not Called** 28508 Called Not Called** Called Not Called** 28511 Called Not Called** Called Not Called** 12988 Not Called Not Called** Not Called Not Called** 13427 Called Not Called** Called Not Called** 41603 Called Not Called** Called Not Called** 28601 Called Not Called** Called Not Called** 6252 Called Not Called** Called Not Called** 6239 Called Not Called** Called Not Called** 12373 Not Called Not Called** Not Called Not Called** 22992 Called Not Called** Called Not Called** 28510 Called Not Called** Called Not Called** 13979 Called Not Called** Called Not Called** 13180 Called Called Called Called 53194 Called Called Called Called 18419 Called Called Called Called 13182 Called Called Called Called 17570 Called Called Called Called 6223 Not Called* Called Not Called* Called 21984 Called Called Called Called 28603 Called Called Called Called 26445 Called Not Called Called Not Called 6241 Called Not Called Called Not Called 12376 Called Not Called Called Not Called 12381 Called Not Called Called Not Called 13007 Called Called Called Called 6240 Called Called Called Called 13192 Called Called Called Called 28610 Called Called Called Called 41663 Called Not Called Called Not Called 6224 Called Not Called Called Not Called 12675 Called Not Called Called Not Called 6213 Called Not Called Called Not Called 14070 Called Not Called Called Not Called 28607 Called Not Called Called Not Called 33725 Called Not Called Called Not Called 13008 Called Not Called Called Not Called 26438 Called Not Called Called Not Called *Mutation not called by software, but manual inspection revealed that the sequence corresponded to the correct mutation **Variant introduced in primer region of test method Called: sequence variant noted by analysis software Not Targeted: sequence variant not included in sequence analyzed by the test method

TABLE 8 Mutation Gene CDS 2.8% 2.8% 5.4% 5.4% 11.0% 11.0% 18.4% 18.4% 29.5% 29.5% 47.9% 47.9% BRAF c.1359T > C p.P453P SNP 4.7 8.8 7 14.3 15.5 24.3 27.4 39.4 39.3 59.8 58.3 EGFR c.340G > A p.E114K SNP 4.7 7.5 7.8 14.5 15.4 25.3 23.9 38.6 39.3 59 58.8 EGFR c.323G > A p.R108K SNP 5.1 7.8 7.9 15 14.8 26.7 25.3 40.4 39.3 59 59.2 BRAF c.1380A > G p.G460G SNP 4.8 8.7 7 14.9 15.5 24.2 27.4 39.3 39.2 59.5 57.8 EGFR c.874G > T p.V292L SNP 4.1 8.7 5.5 12.9 14.4 27.9 27.1 41.5 41.4 62.9 58.9 EGFR c.2235_2249 p.E746_ DEL 18.3 16.8 26.2 27.7 41.4 43.6 61.2 59.5 del15 A750 delELREA KRAS c.111 + 1C > T p.? SNP 4.5 8 6.3 15.4 13.4 24.3 24.7 38.2 37.4 58.5 59.7 EGFR c.2429G > A p.G810D SNP 3.1 5.6 8.3 6.7 12.1 14.4 25.3 25.4 36.8 40 55.1 55.7 EGFR c.2612C > G p.A871G SNP 3.1 5.8 8 7.2 13.7 17.4 24.9 27.5 39.6 40.4 61.2 60.8 EGFR c.2203G > A p.G735S SNP 3.4 4.7 7.8 8.5 15.9 16.2 24.8 29.6 39.8 43.5 57.9 60.7 EGFR c.2375T > C p.L792P SNP 3.9 4.2 8.6 5.7 14.6 14.5 22.1 27.2 39.2 41.1 59 59.5 EGFR c.2170G > A p.G724S SNP 3.8 5 8.8 8.6 16.7 16.9 27.9 28.6 43.1 39.1 62 57.6 EGFR c.2588G > A p.G863D SNP 3.3 5.2 8 7.1 13.9 17.4 23.8 26.9 40.4 40.3 61.6 60.5 KRAS c.104C > T p.T35I SNP 3.9 3.7 7.8 6.9 15.4 13.6 24.4 25.8 38.5 37.4 57.7 59.8 EGFR c.2222C > T p.P741L SNP 3.2 4.6 7.8 7.9 16.2 16.5 25.8 28.1 41.4 43.5 59.1 60.2 KRAS c.351A > C p.K117N SNP 5.2 4.2 7.6 7.1 14.7 15.3 23 24.4 38.3 46.5 55.4 61.2 EGFR c.866C > T p.A289V SNP 3 4.2 9.3 5.6 12.8 14.6 27.9 27 44.2 41.4 62.9 57.6 EGFR c.1793G > T p.G598V SNP 4.2 5.5 7 6.6 14.3 16.1 23.6 24.7 38 41.8 54.8 58.3 EGFR c.2293G > A p.V765M SNP 3.9 4.3 8.8 5.5 14.6 14.3 22.3 27.6 39.3 40.8 58.9 59.9 EGFR c.2441T > C p.L814P SNP 3.1 6.6 9.3 6.8 12 16.2 26.1 25.8 37.5 41.5 55.6 58.7 EGFR c.2092G > A p.A698T SNP 3.7 5.2 9.1 8.9 16.6 17.1 28.2 28.8 41 38.5 62.5 58.5 BRAF c.1391G > T p.G464V SNP 2.7 4.6 8.7 7 14.5 15.4 24.1 27 39.4 39.2 59.4 57.8 BRAF c.1742A > G p.N581S SNP 3.7 4.9 8.9 8.2 12.4 25.1 28.6 39.3 43.9 59.4 57.2 BRAF c.1781A > G p.D594G SNP 4 4.8 8.3 8.4 12.3 25.7 28.4 39.7 43.8 58.2 57.2 BRAF c.1790T > G p.L597R SNP 3.6 4.7 8.1 8.3 12.3 25.9 28.6 38.6 44 57.1 57.4 BRAF c.1799T > A p.V600E SNP 4 4.7 8.6 8.3 12.2 26.4 28.6 39 43.9 58 57.4 KRAS c.24A > G p.V8V SNP 4.7 3 8.3 6.7 14.8 13.2 25 24.3 37 36.7 56.2 59.1 KRAS c.35G > A p.G12D SNP 4.6 3.4 8.1 6.8 15.2 12.8 24.5 24.5 36 36.7 57.4 60 EGFR c.2197C > T p.P733S SNP 3.5 5.2 8 7.5 16 16.8 24.8 28.1 40.9 43.6 59 60.1 KRAS c.175G > A p.A59T SNP 4.5 4.4 8.3 6.5 15.6 14.2 24 26.4 38.1 40.9 58.6 60.7 KRAS c.183A > C p.Q61H SNP 4.5 4.5 7.5 7 15.5 14.3 23.2 26.9 40.3 40.8 60.5 61 EGFR c.2582T > A p.L861Q SNP 2.7 5.7 8.2 6.7 13.8 17.2 23.8 27.1 39.9 40.2 60.6 60.4 EGFR c.2573T > G p.858R SNP 2.7 5.3 8.4 7.1 13.8 17.1 22.9 26 39.8 40.1 60.7 60.6 EGFR c.2156G > C p.G719A SNP 3.6 5.4 9.3 8.4 16.6 16.5 27.9 29.3 42.5 38.7 62.4 57.8 BRAF c.1330C > T p.R444W SNP 2.9 4.5 8.6 6.7 14.5 15.6 24.4 27.9 38 39.2 60.2 59.3

TABLE 9 Chromosome Position Reference Alternate Length Mutation ID Gene Mutation CDS 1 43814979 G A 1 COSM27286 MPL c.1514G > A 1 43815009 G T 1 COSM18918 MPL c.1544G > T 1 43815020 G A 1 COSM27290 MPL c.1555G > A 1 115256529 T C 1 COSM584 NRAS c.182A > G 1 115256537 T C 1 COSM1332933 NRAS c.174A > G 1 115256669 G A 1 gDNA31 NRAS c.112 − 70C > T 1 115258730 C T 1 COSM577 NRAS c.52G > A 1 115258747 C T 1 COSM564 NRAS c.35G > A 1 115258753 C T 1 COSM24850 NRAS c.29G > A 2 29432664 C T 1 COSM28056 ALK c.3824G > A 2 29443695 G T 1 COSM28055 ALK c.3522C > A 2 48030632 G T 1 COSM13399 MSH6 c.3246G > T 2 48030639 AC A 1 COSM13395 MSH6 c.3261delC 2 48030686 G A 1 COSM1021299 MSH6 c.3300G > A 2 48030838 A T 1 gDNA32 MSH6 c.3438 + 14A > T 2 209113112 C T 1 COSM28746 IDH1 c.395G > A 2 209113119 T C 1 COSM1404902 IDH1 c.388A > G 2 209113140 C T 1 COSM96922 IDH1 c.367G > A 2 212288955 C A 1 COSM48362 ERBB4 c.2791G > T 2 212288964 C A 1 COSM169572 ERBB4 c.2782G > T 2 212530084 C T 1 COSM232263 ERBB4 c.1835G > A 2 212530091 G T 1 COSM573362 ERBB4 c.1828C > A 2 212530135 T C 1 COSM1405173 ERBB4 c.1784A > G 2 212576810 A G 1 COSM1614287 ERBB4 c.1089T > C 2 212576877 G A 1 COSM110095 ERBB4 c.1022C > T 2 212576896 C A 1 COSM573356 ERBB4 c.1003G > T 2 212578348 A G 1 COSM1405181 ERBB4 c.909T > C 2 212578372 A C 1 COSM160825 ERBB4 c.885T > G 2 212587172 G T 1 COSM1015994 ERBB4 c.829C > A 2 212587197 G T 1 COSM1251447 ERBB4 c.804C > A 2 212589812 T C 1 COSM1405184 ERBB4 c.730A > G 2 212589838 G A 1 COSM573353 ERBB4 c.704C > T 2 212589909 C T 1 COSM1015997 ERBB4 c.633G > A 2 212652764 T C 1 COSM48369 ERBB4 c.542A > G 2 212652791 G C 1 COSM442267 ERBB4 c.515C > G 3 10183797 T A 1 COSM14305 VHL c.266T > A 3 10183808 G C 1 COSM18080 VHL c.277G > C 3 10183817 C T 1 COSM17658 VHL c.286C > T 3 10183824 AC A 1 COSM17886 VHL c.296delC 3 10188200 C A 1 COSM17752 VHL c.343C > A 3 10188210 T C 1 COSM14312 VHL c.353T > C 3 10188245 G C 1 COSM14407 VHL c.388G > C 3 10188286 CG C 1 COSM14412 VHL c.431delG 3 10191479 C G 1 COSM17657 VHL c.472C > G 3 10191488 C T 1 COSM17612 VHL c.481C > T 3 10191506 C T 1 COSM14311 VHL c.499C > T 3 10191513 T C 1 COSM17837 VHL c.506T > C 3 37067240 T A 1 COSM26085 MLH1 c.1151T > A 3 41266101 C G 1 COSM5677 CTNNB1 c.98C > G 3 41266113 C T 1 COSM5662 CTNNB1 c.110C > T 3 41266124 A G 1 COSM5664 CTNNB1 c.121A > G 3 41266137 C T 1 COSM5667 CTNNB1 c.134C > T 3 138665163 G C 1 COSM33661 FOXL2 c.402C > G 3 178916648 G A 1 COSM27495 PIK3CA c.35G > A 3 178916706 A G 1 COSM27376 PIK3CA c.93A > G 3 178916793 A G 1 COSM1420738 PIK3CA c.180A > G 3 178916823 C T 1 COSM1041454 PIK3CA c.210C > T 3 178916936 G A 1 COSM27497 PIK3CA c.323G > A 3 178916944 A G 1 COSM13570 PIK3CA c.331A > G 3 178916957 G T 1 COSM125368 PIK3CA c.344G > T 3 178917661 A G 1 COSM1420774 PIK3CA c.536A > G 3 178921489 C T 1 COSM21462 PIK3CA c.971C > T 3 178921520 C T 1 COSM353193 PIK3CA c.1002C > T 3 178921553 T A 1 COSM754 PIK3CA c.1035T > A 3 178927450 T C 1 COSM1420804 PIK3CA c.1213T > C 3 178927980 T C 1 COSM757 PIK3CA c.1258T > C 3 178928092 A G 1 COSM1420828 PIK3CA c.1370A > G 3 178936074 C G 1 COSM759 PIK3CA c.1616C > G 3 178936082 G A 1 COSM760 PIK3CA c.1624G > A 3 178936091 G A 1 COSM763 PIK3CA c.1633G > A 3 178936098 A G 1 COSM1420865 PIK3CA c.1640A > G 3 178938860 A C 1 COSM778 PIK3CA c.2102A > C 3 178947827 G T 1 COSM769 PIK3CA c.2702G > T 3 178947850 T C 1 COSM770 PIK3CA c.2725T > C 3 178952055 A G 1 COSM328026 PIK3CA c.3110A > G 3 178952085 A G 1 COSM775 PIK3CA c.3140A > G 3 178952149 C CA 1 COSM12464 PIK3CA c.3204_3205insA 4 1803568 C G 1 COSM715 FGFR3 c.746C > G 4 1803575 C T 1 COSM29446 FGFR3 c.753C > T 4 1803668 GC G 1 COSM723 FGFR3 c.850delC 4 1806089 G T 1 COSM716 FGFR3 c.1108G > T 4 1806119 G A 1 COSM24842 FGFR3 c.1138G > A 4 1806131 T C 1 COSM724 FGFR3 c.1150T > C 4 1806153 C A 1 COSM721 FGFR3 c.1172C > A 4 1807869 A G 1 COSM1428724 FGFR3 c.1928A > G 4 1807889 A G 1 COSM719 FGFR3 c.1948A > G 4 1807894 G A 1 gDNA1 FGFR3 c.1959A > G 4 1808331 G T 1 COSM24802 FGFR3 c.2089G > T 4 1808962 A C 1 AMXsynt1 FGFR3 c.2401A > C 4 55141051 GCCCAG G 15 COSM12418 PDGFRA c.1698_1712del15 ATGGACA TGA 4 55141055 A G 1 gDNA2, PDGFRA c.1701A > G COSM1430082 4 55141097 T C 1 COSM1430085 PDGFRA c.1743T > C 4 55144148 C A 1 COSM22415 PDGFRA c.1977C > A 4 55144172 A G 1 COSM1430086 PDGFRA c.2001A > G 4 55144547 C T 1 COSM743 PDGFRA c.2021C > T 4 55151958 T TA 1 gDNA33 PDGFRA c.2440 − 50_c.2440 − 49insA 4 55152085 G T 1 COSM587613 PDGFRA c.2517G > T 4 55152093 A T 1 COSM736 PDGFRA c.2525A > T 4 55152112 C A 1 COSM28052 PDGFRA c.2544C > A 4 55561702 C T 1 COSM77973 KIT c.92C > T 4 55561764 G A 1 COSM1146 KIT c.154G > A 4 55561828 A G 1 COSM1430106 KIT c.218A > G 4 55592081 T C 1 COSM24637 KIT c.1405T > C 4 55592092 A G 1 COSM41602 KIT c.1416A > G 4 55592178 C CTGCCTA 6 COSM1326 KIT c.1509_1510insGCCTAT 4 55592192 T C 1 COSM96867 KIT c.1516T > C 4 55592202 A T 1 COSM96885 KIT c.1526A > T 4 55592211 A G 1 COSM1430136 KIT c.1535A > G 4 55593431 G A 1 COSM1155 KIT c.1588G > A 4 55593464 A C 1 gDNA3, KIT c.1621A > C COSM28026 4 55593632 C T 1 COSM1275 KIT c.1698C > T 4 55593661 T C 1 COSM1290 KIT c.1727T > C 4 55593689 C T 1 COSM1299 KIT c.1755C > T 4 55594221 A G 1 COSM1304 KIT c.1924A > G 4 55594258 T C 1 COSM12706 KIT c.1961T > C 4 55595599 C T 1 COSM36053 KIT c.2089C > T 4 55597500 T C 1 COSM1430171 KIT c.2148T > C 4 55597561 G A 1 COSM21303 KIT c.2209G > A 4 55599284 C T 1 COSM20402 KIT c.2410C > T 4 55599401 T A 1 COSM19194 KIT c.2484 + 43T > A 4 55599436 T C 1 gDNA4 KIT c.2484 + 78T > C 4 55602737 G A 1 COSM133767 KIT c.2558G > A 4 55602765 G C 1 gDNA5, KIT c.2586G > C COSM1325 4 55946081 A G 1 gDNA6 KDR c.*27T > C 4 55946171 G A 1 COSM35855 KDR c.4008C > T 4 55953816 ACTTCCTC A 25 AMXsynt2 KDR c.3594del25 CTCCTCCA TACAGGAAAC 4 55955112 C T 1 COSM1430203 KDR c.3433G > A 4 55961023 C A 1 COSM48464 KDR c.2917G > T 4 55962505 T C 1 COSM1430212 KDR c.2619A > G 4 55962545 T TG 1 gDNA7 KDR c.2615 − 37_2615 − 36insC 4 55972974 T A 1 gDNA8, KDR c.1416A > T COSM149673 4 55972978 CTATAAGAA C 41 AMXsynt3 KDR c.1413-42del41 AGAGATAAC AGCGCATAT TATGATTTA ATTTTT 4 55979623 C A 1 COSM32339 KDR c.824G > T 4 55980239 C T 1 gDNA9 KDR c.798 + 54G > A 4 153244078 T C 1 COSM1427592 FBXW7 c.2079A > G 4 153244092 G A 1 COSM27083 FBXW7 c.2065C > T 4 153244124 G C 1 COSM732399 FBXW7 c.2033C > G 4 153244155 TC T 1 COSM34018 FBXW7 c.2001delG 4 153245477 T TGATCATAT 31 AMXsynt4 FBXW7 c.1473_1474insCTC TCATATTCT GTTGATTTCAGAGAATA CTGAAATCA TGAATATGATC ACGAG 4 153247222 T C 1 COSM27913 FBXW7 c.1580A > G 4 153247226 A G 1 COSM30599 FBXW7 c.1576T > C 4 153247244 C T 1 COSM30598 FBXW7 c.1558G > A 4 153247351 C A 1 COSM34016 FBXW7 c.1451G > T 4 153247366 C T 1 COSM22974 FBXW7 c.1436G > A 4 153249384 C T 1 COSM22965 FBXW7 c.1394G > A 4 153249440 C T 1 COSM22986 FBXW7 c.1338G > A 4 153249456 C A 1 COSM161024 FBXW7 c.1322G > T 4 153250883 G A 1 COSM22973 FBXW7 c.1177C > T 4 153258983 G A 1 COSM22971 FBXW7 c.832C > T 4 153259071 C A 1 COSM1052125 FBXW7 c.744G > T 5 112173830 G GA 1 COSM18979 APC c.2543_2544insA 5 112173917 C T 1 COSM18852 APC c.2626C > T 5 112173930 T C 1 COSM19230 APC c.2639T > C 5 112173947 C T 1 COSM19330 APC c.2656C > T 5 112174043 G T 1 COSM19065 APC c.2752G > T 5 112174577 C T 1 COSM13872 APC c.3286C > T 5 112174596 A G 1 COSM1432250 APC c.3305A > G 5 112174726 A G 1 COSM1432260 APC c.3435A > G 5 112174989 CA C 1 COSM41617 APC c.3700delA 5 112175086 A G 1 COSM1432280 APC c.3795A > G 5 112175162 C T 1 COSM19072 APC c.3871C > T 5 112175171 C T 1 COSM18960 APC c.3880C > T 5 112175211 T TA 1 COSM18719 APC c.3923_3924insA 5 112175255 G T 1 COSM18702 APC c.3964G > T 5 112175348 G T 1 COSM19048 APC c.4057G > T 5 112175354 T C 1 COSM19652 APC c.4063T > C 5 112175423 C T 1 COSM18862 APC c.4132C > T 5 112175432 C T 1 COSM143913 APC c.4141C > T 5 112175479 TGA T 2 COSM18993 APC c.4189_4190delGA 5 112175507 C T 1 COSM19087 APC c.4216C > T 5 112175576 C T 1 COSM18836 APC c.4285C > T 5 112175675 AAG A 2 COSM13864 APC c.4393_4394delAG 5 112175830 GC G 1 COSM1173082 APC c.4540delC 5 112175852 G T 1 COSM1183180 APC c.4561G > T 5 112175930 G T 1 COSM13879 APC c.4639G > T 5 112175945 G T 1 COSM41616 APC c.4654G > T 5 112175951 G GA 1 COSM18561 APC c.4666_4667insA 5 112176063 C CA 1 COSM18875 APC c.4773_4774insA 5 112176117 C T 1 COSM42906 APC c.4826C > T 5 149433596 T G 1 gDNA10 CSF1R c.*36A > C 5 149433597 G A 1 gDNA11 CSF1R c.*35C > T 5 149433645 T C 1 COSM947 CSF1R c.2906A > G 5 149433673 C T 1 COSM310349 CSF1R c.2878G > A 5 149453057 C CACTGCTTGA 9 AMXsynt5 CSF1R c.890 − 2_c.890 − 1ins TCAAGCAGT 5 170837543 C CTCTG 4 COSM17559 NPM1 c.863_864insTCTG 7 55211080 G A 1 COSM21683 EGFR c.323G > A 7 55211097 G A 1 COSM174732 EGFR c.340G > A 7 55211165 C T 1 COSM1451540 EGFR c.408C > T 7 55221822 C T 1 COSM21687 EGFR c.866C > T 7 55221830 G T 1 COSM43067 EGFR c.874G > T 7 55233043 G T 1 COSM21690 EGFR c.1793G > T 7 55233109 G A 1 COSM35825 EGFR c.1859G > A 7 55241615 T C 1 COSM13177 EGFR c.2063T > C 7 55241644 G A 1 COSM41905 EGFR c.2092G > A 7 55241708 G C 1 COSM6239 EGFR c.2156G > C 7 55241722 G A 1 COSM13979 EGFR c.2170G > A 7 55241755 G A 1 gDNA15 EGFR c.2184 + 19G > A 7 55242427 C T 1 COSM53194 EGFR c.2197C > T 7 55242433 G A 1 COSM13182 EGFR c.2203G > A 7 55242452 C T 1 COSM17570 EGFR c.2222C > T 7 55242464 AGGAATT A 15 COSM6223 EGFR c.2235_2249del15 AAGAGAAGC 7 55248995 G A 1 COSM28603 EGFR c.2293G > A 7 55249063 G A 1 gDNA16, EGFR c.2361G > A COSM1451600 7 55249077 T C 1 COSM13190 EGFR c.2375T > C 7 55249131 G A 1 COSM12986 EGFR c.2429G > A 7 55249143 T C 1 COSM28610 EGFR c.2441T > C 7 55259427 G A 1 COSM53291 EGFR c.2485G > A 7 55259439 T G 1 COSM13424 EGFR c.2497T > G 7 55259446 A T 1 COSM6227 EGFR c.2504A > T 7 55259457 G A 1 COSM13430 EGFR c.2515G > A 7 55259515 T G 1 COSM6224 EGFR c.2573T > G 7 55259524 T A 1 COSM6213 EGFR c.2582T > A 7 55259530 G A 1 COSM14070 EGFR c.2588G > A 7 55259554 C G 1 COSM13008 EGFR c.2612C > G 7 116339642 G T 1 COSM706 MET c.504G > T 7 116340262 A G 1 COSM710 MET c.1124A > G 7 116412044 G A 1 COSM29633 MET c.3082 + 1G > A 7 116417465 T C 1 COSM1447462 MET c.3336T > C 7 116417499 C G 1 COSM697 MET c.3370C > G 7 116418969 G C 1 COSM43064 MET c.3534G > C 7 116418997 C T 1 COSM1214928 MET c.3562C > T 7 116419008 T C 1 COSM1447471 MET c.3573T > C 7 116422133 T G 1 COSM1330154 MET c.3668T > G 7 116423428 T G 1 COSM700 MET c.3757T > G 7 116423449 G T 1 COSM48565 MET c.3778G > T 7 116423456 A G 1 COSM695 MET c.3785A > G 7 116423474 T C 1 COSM691 MET c.3803T > C 7 116436022 G A 1 gDNA12, MET c.4071G > A COSM150378 7 116436097 G A 1 gDNA13, MET c.4146G > A COSM150379 7 128845101 C T 1 COSM13145 SMO c.595C > T 7 128846040 G A 1 COSM13147 SMO c.970G > A 7 128846398 C T 1 COSM216037 SMO c.1234C > T 7 128846469 A G 1 gDNA14 SMO c.1264 + 41A > G 7 128850341 G T 1 COSM13146 SMO c.1604G > T 7 128851593 A G 1 COSM13150 SMO c.1918A > G 7 140453136 A T 1 COSM476 BRAF c.1799T > A 7 140453145 A C 1 COSM471 BRAF c.1790T > G 7 140453154 T C 1 COSM467 BRAF c.1781A > G 7 140453193 T C 1 COSM462 BRAF c.1742A > G 7 140481417 C A 1 COSM450 BRAF c.1391G > T 7 140481428 T C 1 COSM27986 BRAF c.1380A > G 7 140481449 A G 1 COSM1448625 BRAF c.1359T > C 7 140481478 G A 1 COSM6262 BRAF c.1330C > T 7 148508727 T A 1 COSM37028 EZH2 c.1937A > T 8 38282147 G A 1 COSM1292693 FGFR1 c.816C > T 8 38285864 G A 1 COSM187237 FGFR1 c.448C > T 8 38285891 T C 1 COSM1456955 FGFR1 c.421A > G 8 38285938 G A 1 COSM601 FGFR1 c.374C > T 9 5073770 G T 1 COSM12600 JAK2 c.1849G > T 9 5073781 C A 1 COSM27063 JAK2 c.1860C > A 9 21971000 C A 1 COSM12479 CDKN2A c.358G > T 9 21971017 G A 1 COSM12476 CDKN2A c.341C > T 9 21971028 C T 1 COSM12547 CDKN2A c.330G > A 9 21971036 C A 1 COSM13489 CDKN2A c.322G > T 9 21971111 G A 1 COSM12504 CDKN2A c.247C > T 9 21971120 G A 1 COSM12475 CDKN2A c.238C > T 9 21971153 C A 1 COSM13281 CDKN2A c.205G > T 9 21971186 G A 1 COSM12473 CDKN2A c.172C > T 9 80336317 G A 1 COSM1110323 GNAQ c.1002C > T 9 80343489 T TGTAC 4 AMXsynt6 GNAQ c.829_830insGTAC 9 80343605 GAAAAAACAAGGA G 12 AMXsynt7 GNAQ c.736 − 34del12 9 80409345 A G 1 gDNA17 GNAQ c.735 + 34T > C 9 80409432 ACA TGC 3 AMXsynt8 GNAQ c.679_681TGT > GCA 9 80409533 TAATAACATA T 41 AMXsynt9 GNAQ c.606 − 66del41 TAAAGTAAAA CTAAAAAGTC AACATAAATATA 9 80412493 C T 1 COSM52975 GNAQ c.548G > A 9 80412518 T A 1 COSM1463119 GNAQ c.523A > T 9 133738342 C G 1 COSM12631 ABL1 c.742C > G 9 133738349 G A 1 COSM12577 ABL1 c.749G > A 9 133738357 T C 1 COSM12576 ABL1 c.757T > C 9 133738363 G A 1 COSM12573 ABL1 c.763G > A 9 133747520 A G 1 COSM12602 ABL1 c.827A > G 9 133747571 T TGCC 3 COSM235737 ABL1 c.878_879insGCC 9 133748391 T C 1 COSM12578 ABL1 c.1052T > C 9 133748403 A G 1 COSM12611 ABL1 c.1064A > G 9 133748414 T G 1 COSM12605 ABL1 c.1075T > G 9 133750319 C A 1 COSM49071 ABL1 c.1150C > A 9 133750356 A G 1 COSM12604 ABL1 c.1187A > G 9 139390779 G T 1 COSM87862 NOTCH1 c.7412C > A 9 139390804 CG C 1 COSM13070 NOTCH1 c.7386delC 9 139390816 G A 1 COSM12776 NOTCH1 c.7375C > T 9 139390873 G A 1 COSM13061 NOTCH1 c.7318C > T 9 139397768 A G 1 COSM13048 NOTCH1 c.5033T > C 9 139397776 G A 1 COSM308587 NOTCH1 c.5025C > T 9 139399344 A G 1 COSM12771 NOTCH1 c.4799T > C 9 139399350 C G 1 COSM13053 NOTCH1 c.4793G > C 9 139399365 A G 1 COSM13042 NOTCH1 c.4778T > C 9 139399422 A G 1 COSM12772 NOTCH1 c.4721T > C 10 43609096 T C 1 COSM29803 RET c.1852T > C 10 43609102 T C 1 COSM29804 RET c.1858T > C 10 43609942 GAGCTGTGCCGCA AGCT 13 COSM1048 RET c.1894_1906 > AGCT 10 43609990 G A 1 COSM1223553 RET c.1942G > A 10 43610039 C A 1 COSM976 RET c.1991C > A 10 43613840 G C 1 COSM21338 RET c.2304G > C 10 43613843 G T 1 gDNA18 RET c.2307T > A 10 43615568 GC TT 3 COSM977 RET c.2647_2648GC > TT 10 43615622 G A 1 COSM963 RET c.2701G > A 10 43617416 T C 1 COSM965 RET c.2753T > C 10 89624245 G T 1 COSM5298 PTEN c.19G > T 10 89624266 A G 1 COSM5101 PTEN c.40A > G 10 89624275 C T 1 COSM5153 PTEN c.49C > T 10 89624297 A G 1 COSM5107 PTEN c.71A > G 10 89653686 A G 1 gDNA34 PTEN c.80 − 96A > G 10 89653782 A G 1 COSM5134 PTEN c.80A > G 10 89653814 C T 1 COSM5142 PTEN c.112C > T 10 89653844 A G 1 COSM5050 PTEN c.142A > G 10 89653858 T C 1 COSM1349479 PTEN c.156T > C 10 89653865 A G 1 COSM5129 PTEN c.163A > G 10 89685271 T G 1 COSM5257 PTEN c.166T > G 10 89685307 T C 1 COSM5036 PTEN c.202T > C 10 89685319 G A 1 COSM5916 PTEN c.209 + 5G > A 10 89690805 G A 1 COSM5102 PTEN c.212G > A 10 89690818 TTA T 2 COSM4956 PTEN c.227_228delAT 10 89690838 A C 1 COSM5205 PTEN c.245A > C 10 89690847 G A 1 COSM5983 PTEN c.253 + 1G > A 10 89692779 A G 1 COSM5139 PTEN c.263A > G 10 89692818 T C 1 COSM5109 PTEN c.302T > C 10 89692830 G T 1 COSM5266 PTEN c.314G > T 10 89692850 C G 1 COSM5199 PTEN c.334C > G 10 89692911 G A 1 COSM5123 PTEN c.395G > A 10 89692965 A G 1 COSM5130 PTEN c.449A > G 10 89692980 A G 1 COSM5144 PTEN c.464A > G 10 89692993 G T 1 COSM5287 PTEN c.477G > T 10 89711855 CT C 1 COSM5907 PTEN c.493 − 12delT 10 89711960 T C 1 COSM35406 PTEN c.578T > C 10 89711972 AGAT A 3 COSM4978 PTEN c.595_597delATG 10 89711992 C A 1 COSM5279 PTEN c.610C > A 10 89711997 G A 1 COSM5072 PTEN c.615G > A 10 89717672 C T 1 COSM5154 PTEN c.697C > T 10 89717678 G T 1 COSM5292 PTEN c.703G > T 10 89717696 T C 1 COSM35849 PTEN c.721T > C 10 89717762 A T 1 COSM43075 PTEN c.787A > T 10 89717769 TA T 1 COSM5809 PTEN c.800delA 10 89720728 A G 1 COSM1349606 PTEN c.879A > G 10 89720744 G T 1 COSM5312 PTEN c.895G > T 10 89720798 GTACT G 4 COSM4958 PTEN c.955_958delACTT 10 89720874 A G 1 COSM1349625 PTEN c.1025A > G 10 89720907 T G 1 gDNA19 PTEN c.1026 + 32T > G 10 89725042 A G 1 COSM5966 PTEN c.1027 − 2A > G 10 89725056 TTC T 2 COSM4936 PTEN c.1040_1041delTC 10 89725072 A G 1 COSM1349633 PTEN c.1055A > G 10 89725108 C G 1 COSM23645 PTEN c.1091C > G 10 123258034 A T 1 COSM36912 FGFR2 c.1647T > A 10 123274774 A G 1 COSM36906 FGFR2 c.1144T > C 10 123274794 T C 1 COSM36904 FGFR2 c.1124A > G 10 123274810 T C 1 COSM1346272 FGFR2 c.1108A > G 10 123279503 T C 1 COSM36901 FGFR2 c.929A > G 10 123279519 C T 1 COSM29824 FGFR2 c.913G > A 10 123279677 G C 1 COSM36903 FGFR2 c.755C > G 11 533874 T C 1 COSM499 HRAS c.182A > G 11 533881 C T 1 COSM495 HRAS c.175G > A 11 534242 A G 1 gDNA20, HRAS c.81T > C COSM249860 11 534288 C A 1 COSM483 HRAS c.35G > T 11 108117798 C T 1 COSM21323 ATM c.1009C > T 11 108119823 T C 1 COSM21825 ATM c.1229T > C 11 108123551 C T 1 COSM22499 ATM c.1810C > T 11 108123641 T A 1 COSM1158828 ATM c.1898 + 2T > A 11 108138003 T C 1 COSM21826 ATM c.2572T > C 11 108155132 G A 1 COSM22507 ATM c.3925G > A 11 108170479 G T 1 COSM21920 ATM c.5044G > T 11 108170587 C G 1 COSM218294 ATM c.5152C > G 11 108172374 G T 1 COSM49005 ATM c.5178 − 1G > T 11 108172385 C T 1 COSM172204 ATM c.5188C > T 11 108172421 G C 1 COSM21918 ATM c.5224G > C 11 108173640 C T 1 COSM12792 ATM c.5380C > T 11 108173736 T G 1 COSM1183962 ATM c.5476T > G 11 108180945 G C 1 COSM21922 ATM c.5821G > C 11 108200958 A C 1 COSM12951 ATM c.7325A > C 11 108204681 A G 1 COSM12791 ATM c.7996A > G 11 108205769 G C 1 COSM21636 ATM c.8084G > C 11 108205780 C A 1 COSM1235404 ATM c.8095C > A 11 108206594 A T 1 COSM22481 ATM c.8174A > T 11 108218045 A G 1 COSM1183939 ATM c.8624A > G 11 108218089 C G 1 COSM22485 ATM c.8668C > G 11 108218196 T C 1 gDNA21 ATM c.8671 + 104T > C 11 108225590 A T 1 COSM21930 ATM c.8839A > T 11 108225661 A G 1 gDNA22 ATM c.8850 + 60A > G 11 108236087 G A 1 COSM21626 ATM c.9023G > A 11 108236118 A G 1 COSM1351060 ATM c.9054A > G 11 108236203 C T 1 COSM21624 ATM c.9139C > T 12 25362805 C T 1 COSM41307 KRAS c.491G > A 12 25378647 T G 1 COSM19940 KRAS c.351A > C 12 25380275 T G 1 COSM554 KRAS c.183A > C 12 25380283 C T 1 COSM546 KRAS c.175G > A 12 25398207 C A 1 AMXsynt11 KRAS c.111 + 1C > T 12 25398215 G A 1 COSM14208 KRAS c.104C > T 12 25398284 C T 1 COSM521 KRAS c.35G > A 12 25398295 T C 1 COSM507 KRAS c.24A > G 12 112888165 G T 1 COSM13011 PTPN11 c.181G > T 12 112888189 G A 1 COSM13013 PTPN11 c.205G > A 12 112888199 C T 1 COSM13015 PTPN11 c.215C > T 12 112888210 G A 1 COSM13000 PTPN11 c.226G > A 12 112926852 C T 1 COSM13034 PTPN11 c.1472C > T 12 112926888 G C 1 COSM13027 PTPN11 c.1508G > C 12 112926899 A G 1 COSM1358900 PTPN11 c.1519A > G 12 112926908 C A 1 COSM13031 PTPN11 c.1528C > A 12 121431413 G T 1 COSM21471 HNF1A c.617G > T 12 121431428 A C 1 COSM24900 HNF1A c.632A > C 12 121431481 C T 1 COSM24832 HNF1A c.685C > T 12 121431506 A G 1 COSM21474 HNF1A c.710A > G 12 121432032 C T 1 COSM24923 HNF1A c.779C > T 12 121432040 C T 1 COSM24692 HNF1A c.787C > T 12 121432117 G GC 1 COSM21481 HNF1A c.872_873insC 12 121432117 G C 1 gDNA23 HNF1A c.864G > C 13 28592629 T C 1 COSM1166729 FLT3 c.2516A > G 13 28592642 C A 1 COSM783 FLT3 c.2503G > T 13 28592653 C T 1 COSM25248 FLT3 c.2492G > A 13 28602329 G A 1 COSM786 FLT3 c.2039C > T 13 28608255 G GATCATAT 21 COSM27907 FLT3 c.1800_1801ins TCATATTC TTCAGAGAATATGA TCTGAA ATATGAT 13 28608281 A G 1 COSM19522 FLT3 c.1775T > C 13 28610138 G A 1 COSM28042 FLT3 c.1352C > T 13 28610183 A G 1 gDNA24 FLT3 c.1310 − 3T > C 13 48919244 G T 1 COSM890 RB1 c.409G > T 13 48923148 T A 1 COSM915 RB1 c.596T > A 13 48941628 A T 1 COSM28816 RB1 c.940 − 2A > T 13 48941648 C T 1 COSM891 RB1 c.958C > T 13 48941658 A G 1 COSM1367204 RB1 c.968A > G 13 48941672 A G 1 COSM1367206 RB1 c.982A > G 13 48942685 C T 1 COSM879 RB1 c.1072C > T 13 48953760 C T 1 COSM895 RB1 c.1363C > T 13 48955538 C T 1 COSM887 RB1 c.1654C > T 13 48955550 C T 1 COSM888 RB1 c.1666C > T 13 48955571 T C 1 COSM1367255 RB1 c.1687T > C 13 49027168 C T 1 COSM892 RB1 c.1735C > T 13 49027249 T C 1 COSM35483 RB1 c.1814 + 2T > C 13 49033890 TAGAACA T 13 COSM870 RB1 c.2028_2040del13 TATCATC 13 49033916 C T 1 COSM13117 RB1 c.2053C > T 13 49033926 T C 1 COSM942 RB1 c.2063T > C 13 49037865 A G 1 COSM1042 RB1 c.2107 − 2A > G 13 49037877 G T 1 COSM883 RB1 c.2117G > T 13 49037903 A T 1 COSM940 RB1 c.2143A > T 13 49037913 A G 1 COSM1367309 RB1 c.2153A > G 13 49039164 G T 1 COSM868 RB1 c.2242G > T 13 49039183 T G 1 COSM916 RB1 c.2261T > G 13 49039189 A G 1 COSM551465 RB1 c.2267A > G 13 49039215 A T 1 COSM254910 RB1 c.2293A > T 14 105246551 C T 1 COSM33765 AKT1 c.49G > A 15 66727455 G T 1 COSM1235478 MAP2K1 c.171G > T 15 66727483 G A 1 COSM1235479 MAP2K1 c.199G > A 15 90631838 C T 1 COSM33733 IDH2 c.515G > A 15 90631879 T C 1 COSM1375400 IDH2 c.474A > G 15 90631934 C T 1 COSM41590 IDH2 c.419G > A 16 68835650 GGTGTGAT G 30 AMXsynt10 CDH1 c.241del30 TACAGTCA AAAGGCCTC TACGGT 16 68846087 A G 1 COSM1379165 CDH1 c.1058A > G 16 68846137 G C 1 COSM19748 CDH1 c.1108G > C 16 68847282 G A 1 COSM19750 CDH1 c.1204G > A 16 68855924 A AC 1 COSM25267 CDH1 c.1733_1734insC 16 68855934 T C 1 COSM19746 CDH1 c.1742T > C 16 68855966 G A 1 COSM19758 CDH1 c.1774G > A 16 68856041 G A 1 COSM19743 CDH1 c.1849G > A 16 68856093 C T 1 COSM19822 CDH1 c.1901C > T 16 68856105 G A 1 COSM19418 CDH1 c.1913G > A 17 7572962 GT G 1 COSM13747 TP53 c.1146delA 17 7572986 G A 1 COSM307348 TP53 c.1123C > T 17 7573010 T C 1 COSM1191161 TP53 c.1101 − 2A > G 17 7574003 G A 1 COSM11073 TP53 c.1024C > T 17 7574012 C A 1 COSM11286 TP53 c.1015G > T 17 7574018 G A 1 COSM11071 TP53 c.1009C > T 17 7574026 C A 1 COSM11514 TP53 c.1001G > T 17 7576855 G A 1 COSM11354 TP53 c.991C > T 17 7576865 A C 1 COSM44823 TP53 c.981T > G 17 7576883 T C 1 COSM46088 TP53 c.963A > G 17 7576897 G A 1 COSM10786 TP53 c.949C > T 17 7577022 G A 1 COSM410663 TP53 c.916C > T 17 7577046 C A 1 COSM410710 TP53 c.892G > T 17 7577105 G A 1 COSM410863 TP53 c.833C > T 17 7577120 C T 1 COSM410660 TP53 c.818G > A 17 7577538 C T 1 COSM410662 TP53 c.743G > A 17 7577548 C T 1 COSM46932 TP53 c.733G > A 17 7577559 G A 1 COSM410812 TP53 c.722C > T 17 7577580 T C 1 COSM410725 TP53 c.701A > G 17 7578115 T C 1 gDNA25 TP53 c.672 + 62A > G 17 7578190 T C 1 COSM410758 TP53 c.659A > G 17 7578196 A T 1 COSM44317 TP53 c.653T > A 17 7578203 C T 1 COSM410667 TP53 c.646G > A 17 7578235 T C 1 COSM43947 TP53 c.614A > G 17 7578388 C T 1 COSM410738 TP53 c.542G > A 17 7578442 T C 1 COSM410808 TP53 c.488A > G 17 7578449 C T 1 COSM410739 TP53 c.481G > A 17 7578461 C A 1 COSM410670 TP53 c.469G > T 17 7578526 C T 1 COSM410801 TP53 c.404G > A 17 7578535 T C 1 COSM411582 TP53 c.395A > G 17 7578542 G C 1 COSM411462 TP53 c.388C > G 17 7578550 G A 1 COSM44226 TP53 c.380C > T 17 7579295 C T 1 COSM44985 TP53 c.375 + 17G > A 17 7579312 C T 1 COSM43904 TP53 c.375G > A 17 7579358 C A 1 COSM410716 TP53 c.329G > T 17 7579368 A C 1 COSM46103 TP53 c.319T > G 17 7579414 C T 1 COSM44492 TP53 c.273G > A 17 7579442 G A 1 COSM43910 TP53 c.245C > T 17 7579472 G C 1 gDNA26, TP53 c.215G > C COSM4250061 17 7579521 C A 1 COSM412168 TP53 c.166G > T 17 7579536 C A 1 COSM44907 TP53 c.151G > T 17 7579553 A G 1 COSM43664 TP53 c.134T > C 17 7579575 G A 1 COSM46286 TP53 c.112C > T 17 7579715 AG A 1 COSM485573 TP53 c.80delC 17 7579801 G C 1 gDNA35 TP53 c.74 + 38C > G 17 37880220 T C 1 COSM14060 ERBB2 c.2264T > C 17 37880261 G T 1 COSM1251412 ERBB2 c.2305G > T 17 37880981 A AGCATACGTGATG 12 COSM20959 ERBB2 c.2324_2325ins12 17 37881332 G A 1 COSM14065 ERBB2 c.2524G > A 17 37881378 A G 1 COSM686 ERBB2 c.2570A > G 17 37881440 C T 1 COSM21985 ERBB2 c.2632C > T 18 48575112 T C 1 COSM1389031 SMAD4 c.306T > C 18 48575183 T C 1 COSM14229 SMAD4 c.377T > C 18 48575195 C T 1 COSM218557 SMAD4 c.389C > T 18 48575209 C T 1 COSM14168 SMAD4 c.403C > T 18 48575671 C G 1 COSM13115 SMAD4 c.431C > G 18 48581198 G T 1 COSM14118 SMAD4 c.502G > T 18 48581229 C A 1 COSM1226725 SMAD4 c.533C > A 18 48581243 C T 1 COSM308153 SMAD4 c.547C > T 18 48584560 C T 1 COSM14057 SMAD4 c.733C > T 18 48584593 C T 1 COSM22901 SMAD4 c.766C > T 18 48584602 ACT A 2 COSM14217 SMAD4 c.776_777delCT 18 48586262 C T 1 COSM14163 SMAD4 c.931C > T 18 48586291 G C 1 COSM14167 SMAD4 c.955 + 5G > C 18 48591838 A G 1 COSM1389054 SMAD4 c.1001A > G 18 48591847 A G 1 COSM1389057 SMAD4 c.1010A > G 18 48591855 A G 1 COSM14109 SMAD4 c.1018A > G 18 48591865 C G 1 COSM14111 SMAD4 c.1028C > G 18 48593405 G C 1 COSM14249 SMAD4 c.1156G > C 18 48593465 G A 1 COSM14103 SMAD4 c.1216G > A 18 48593475 T TAC 2 COSM14223 SMAD4 c.1229_1230insCA 18 48593495 A G 1 AMXsynt12 SMAD4 c.1246A > G 18 48603032 C T 1 COSM14096 SMAD4 c.1333C > T 18 48604682 A G 1 COSM14114 SMAD4 c.1504A > G 18 48604697 A G 1 COSM1389099 SMAD4 c.1519A > G 18 48604754 G T 1 COSM14134 SMAD4 c.1576G > T 18 48604769 C A 1 COSM1389106 SMAD4 c.1591C > A 19 1207076 TG T 1 COSM21212 STK11 c.169delG 19 1220321 T C 1 gDNA27 STK11 c.465 − 51T > C 19 1220371 G T 1 COSM21570 STK11 c.465 − 1G > T 19 1220382 C T 1 COSM27316 STK11 c.475C > T 19 1220487 G T 1 COSM20944 STK11 c.580G > T 19 1220502 G T 1 COSM25229 STK11 c.595G > T 19 1221293 C T 1 COSM29005 STK11 c.816C > T 19 1221319 C T 1 COSM21355 STK11 c.842C > T 19 1223125 C G 1 COSM21360 STK11 c.1062C > G 19 3115012 C T 1 COSM21651 GNA11 c.547C > T 19 3118942 A T 1 COSM52969 GNA11 c.626A > T 19 3119239 C T 1 gDNA28 GNA11 c.771C > T 19 17945696 C T 1 COSM34213 JAK3 c.2164G > A 19 17948009 G A 1 COSM34214 JAK3 c.1715C > T 20 36031631 C T 1 COSM1227526 SRC c.1460C > T 20 57480494 C T 1 COSM244725 GNAS c.489C > T 20 57484420 C T 1 COSM27887 GNAS c.601C > T 20 57484596 A T 1 COSM27888 GNAS c.680A > T 22 24133967 C T 1 COSM1002 SMARCB1 c.118C > T 22 24133990 C A 1 COSM991 SMARCB1 c.141C > A 22 24134006 C T 1 COSM24595 SMARCB1 c.157C > T 22 24143240 C T 1 COSM992 SMARCB1 c.472C > T 22 24145528 C CCCGAGG 19 COSM51386 SMARCB1 c.566_567ins19 TGCTGGT CCCCAT 22 24145582 C T 1 COSM993 SMARCB1 c.601C > T 22 24145588 G A 1 COSM999 SMARCB1 c.607G > A 22 24145675 G C 1 gDNA30 SMARCB1 c.601 + 66G > C 22 24176353 GC G 1 COSM1057 SMARCB1 c.1148delC Target Chromo- Mutation Fre- Sanger CHPv2 TSACP TSTP some Position Mutation AA Type Strand quency Detection* Detection Detection Detection 1 43814979 p.S505N SNV + 5-15% Detected Detected Not Covered Not Covered 1 43815009 p.W515L SNV + 5-15% Detected Detected Detected Not Covered 1 43815020 p.A519T SNV + 5-15% Detected Detected Detected Not Covered 1 115256529 p.Q61R SNV − 5-15% Detected Detected Detected Detected 1 115256537 p.T58T SNV − 5-15% Detected Detected Detected Detected 1 115256669 p.(=) SNV − genomic Not Covered Not Covered Not Covered Detected 1 115258730 p.A18T SNV − 5-15% Detected Detected Detected Detected 1 115258747 p.G12D SNV − 5-15% Detected Detected Detected Detected 1 115258753 p.G10E SNV − 5-15% Detected Detected Detected Detected 2 29432664 p.R1275Q SNV − 5-15% Detected Detected Detected Not Covered 2 29443695 p.F1174L SNV − 5-15% Detected Detected Detected Detected 2 48030632 p.P1082P SNV + 5-15% Detected Not Covered Not Covered Detected 2 48030639 p.F1088fs*2 DEL + 5-15% Detected Not Covered Not Covered Detected 2 48030686 p.T1100T SNV + 5-15% Detected Not Covered Not Covered Detected 2 48030838 p.(=) SNV + genomic Not Covered Not Covered Not Covered Detected 2 209113112 p.R132H SNV − 5-15% Detected Detected Detected Not Covered 2 209113119 p.I130V SNV − 5-15% Detected Detected Detected Not Covered 2 209113140 p.G123R SNV − 5-15% Detected Detected Detected Not Covered 2 212288955 p.D931Y SNV − 5-15% Detected Detected Detected Not Covered 2 212288964 p.E928* SNV − 5-15% Detected Detected Detected Not Covered 2 212530084 p.R612Q SNV − 5-15% Detected Detected Detected Not Covered 2 212530091 p.P610T SNV − 5-15% Detected Detected Detected Not Covered 2 212530135 p.D595G SNV − 5-15% Detected Detected Detected Not Covered 2 212576810 p.N363N SNV − 5-15% Detected Detected Not Covered Not Covered 2 212576877 p.S341L SNV − 5-15% Detected Detected Detected Not Covered 2 212576896 p.D335Y SNV − 5-15% Detected Detected Detected Not Covered 2 212578348 p.S303S SNV − 5-15% Detected Detected Detected Not Covered 2 212578372 p.H295Q SNV − 5-15% Detected Detected Detected Not Covered 2 212587172 p.H277N SNV − 5-15% Detected Detected Detected Not Covered 2 212587197 p.Y268* SNV − 5-15% Detected Detected Detected Not Covered 2 212589812 p.T244A SNV − 5-15% Detected Detected Detected Not Covered 2 212589838 p.A235V SNV − 5-15% Detected Detected Detected Not Covered 2 212589909 p.T211T SNV − 5-15% Detected Not Covered Detected Not Covered 2 212652764 p.N181S SNV − 5-15% Detected Detected Detected Not Covered 2 212652791 p.P172R SNV − 5-15% Detected Detected Detected Not Covered 3 10183797 p.L89H SNV + 5-15% Detected Detected Detected Not Covered 3 10183808 p.G93R SNV + 5-15% Detected Detected Detected Not Covered 3 10183817 p.Q96* SNV + 5-15% Detected Detected Detected Not Covered 3 10183824 p.P99fs*60 DEL + 5-15% Detected Detected Detected Not Covered 3 10188200 p.H115N SNV + 5-15% Detected Detected Not Covered Not Covered 3 10188210 p.L118P SNV + 5-15% Detected Detected Not Covered Not Covered 3 10188245 p.V130L SNV + 5-15% Detected Detected Detected Not Covered 3 10188286 p.G144fs*15 DEL + 5-15% Detected Detected Detected Not Covered 3 10191479 p.L158V SNV + 5-15% Detected Detected Not Detected Not Covered 3 10191488 p.R161* SNV + 5-15% Detected Detected Detected Not Covered 3 10191506 p.R167W SNV + 5-15% Detected Detected Detected Not Covered 3 10191513 p.L169P SNV + 5-15% Detected Detected Not Detected Not Covered 3 37067240 p.V384D SNV + 5-15% Detected Detected Detected Not Covered 3 41266101 p.S33C SNV + 5-15% Detected Detected Detected Detected 3 41266113 p.S37F SNV + 5-15% Detected Detected Detected Detected 3 41266124 p.T41A SNV + 5-15% Detected Detected Detected Detected 3 41266137 p.S45F SNV + 5-15% Detected Detected Detected Detected 3 138665163 p.C134W SNV − 5-15% Detected Not Covered Not Covered Detected 3 178916648 p.G12D SNV + 5-15% Detected Not Covered Not Covered Detected 3 178916706 p.I31M SNV + 5-15% Detected Not Covered Not Covered Detected 3 178916793 p.Q60Q SNV + 5-15% Detected Detected Not Covered Detected 3 178916823 p.F7OF SNV + 5-15% Detected Detected Not Covered Detected 3 178916936 p.R108H SNV + 5-15% Detected Detected Detected Detected 3 178916944 p.K111E SNV + 5-15% Detected Detected Detected Detected 3 178916957 p.R115L SNV + 5-15% Detected Detected Detected Detected 3 178917661 p.K179R SNV + 5-15% Detected Not Covered Not Covered Detected 3 178921489 p.T324I SNV + 5-15% Detected Detected Not Covered Not Covered 3 178921520 p.L334L SNV + 5-15% Detected Detected Not Covered Not Covered 3 178921553 p.N345K SNV + 5-15% Detected Detected Detected Not Covered 3 178927450 p.S405P SNV + 5-15% Detected Detected Not Covered Not Covered 3 178927980 p.C420R SNV + 5-15% Detected Detected Detected Detected 3 178928092 p.N457S SNV + 5-15% Detected Detected Not Covered Detected 3 178936074 p.P539R SNV + 5-15% Detected Detected Detected Detected 3 178936082 p.E542K SNV + 5-15% Detected Detected Detected Detected 3 178936091 p.E545K SNV + 5-15% Detected Detected Detected Detected 3 178936098 p.E547G SNV + 5-15% Detected Detected Detected Detected 3 178938860 p.H701P SNV + 5-15% Detected Detected Detected Not Covered 3 178947827 p.C901F SNV + 5-15% Detected Detected Not Covered Not Covered 3 178947850 p.F909L SNV + 5-15% Detected Detected Not Covered Not Covered 3 178952055 p.E1037G SNV + 5-15% Detected Detected Not Detected Detected 3 178952085 p.H1047R SNV + 5-15% Detected Detected Not Detected Detected 3 178952149 p.N1068fs*4 INS + 5-15% Detected Detected Detected Not Detected 4 1803568 p.S249C SNV + 5-15% Detected Detected Detected Not Covered 4 1803575 p.H251H SNV + 5-15% Detected Detected Detected Not Covered 4 1803668 p.H284fs*10 DEL + 5-15% Detected Not Covered Detected Not Covered 4 1806089 p.G370C SNV + 5-15% Detected Detected Not Covered Not Covered 4 1806119 p.G380R SNV + 5-15% Detected Detected Detected Not Covered 4 1806131 p.F384L SNV + 5-15% Detected Detected Detected Not Covered 4 1806153 p.A391E SNV + 5-15% Detected Detected Detected Not Covered 4 1807869 p.H643R SNV + 5-15% Detected Detected Detected Not Covered 4 1807889 p.K650E SNV + 5-15% Detected Detected Detected Not Covered 4 1807894 p.T651T SNV + genomic Not Covered Detected Detected Not Covered 4 1808331 p.G697C SNV + 5-15% Detected Detected Not Detected Not Covered 4 1808962 p.P800P SNV + 5-15% Detected Detected Detected Not Covered 4 55141051 p.5566_E571 > R DEL + 5-15% Detected Detected Detected Detected 4 55141055 p.P567P SNV + genomic Not Covered Detected Detected Detected 4 55141097 p.P581P SNV + 5-15% Detected Detected Detected Detected 4 55144148 p.N659K SNV + 5-15% Detected Detected Detected Detected 4 55144172 p.S667S SNV + 5-15% Detected Detected Detected Detected 4 55144547 p.T674I SNV + 5-15% Detected Detected Detected Not Covered 4 55151958 p.(=) INS + genomic Not Covered Not Covered Not Covered Detected 4 55152085 p.L839L SNV + 5-15% Detected Detected Detected Detected 4 55152093 p.D842V SNV + 5-15% Detected Detected Detected Detected 4 55152112 p.N848K SNV + 5-15% Detected Detected Detected Detected 4 55561702 p.P31L SNV + 5-15% Detected Detected Not Covered Not Covered 4 55561764 p.D52N SNV + 5-15% Detected Detected Detected Not Covered 4 55561828 p.E73G SNV + 5-15% Detected Not Covered Detected Not Covered 4 55592081 p.F469L SNV + 5-15% Detected Not Covered Not Covered Detected 4 55592092 p.L472L SNV + 5-15% Detected Not Covered Not Covered Detected 4 55592178 p.Y503_F504insAY INS + 5-15% Detected Detected Not Covered Detected 4 55592192 p.F506L SNV + 5-15% Detected Detected Detected Detected 4 55592202 p.K509I SNV + 5-15% Detected Detected Detected Detected 4 55592211 p.N512S SNV + 5-15% Detected Detected Detected Detected 4 55593431 p.V530I SNV + 5-15% Detected Detected Detected Not Covered 4 55593464 p.M541L SNV + genomic Detected Detected Not Covered 4 55593632 p.N566N SNV + 5-15% Detected Detected Detected Detected 4 55593661 p.L576P SNV + 5-15% Detected Detected Detected Detected 4 55593689 p.P585P SNV + 5-15% Detected Detected Detected Detected 4 55594221 p.K642E SNV + 5-15% Detected Detected Detected Detected 4 55594258 p.V654A SNV + 5-15% Detected Detected Detected Detected 4 55595599 p.H697Y SNV + 5-15% Detected Not Covered Detected Not Covered 4 55597500 p.D716D SNV + 5-15% Detected Detected Detected Not Covered 4 55597561 p.D7376N SNV + 5-15% Detected Not Covered Detected Not Covered 4 55599284 p.R804W SNV + 5-15% Detected Detected Not Covered Detected 4 55599401 p.? SNV + 5-15% Detected Not Covered Detected Detected 4 55599436 p.(=) SNV + genomic Not Covered Not Covered Detected Not Detected 4 55602737 p.W853* SNV + 5-15% Detected Detected Detected Detected 4 55602765 p.L862L SNV + genomic Not Covered Not Covered Detected Detected 4 55946081 p.(=) SNV − genomic Not Covered Not Covered Detected Not Covered 4 55946171 p.T1336T SNV − 5-15% Detected Detected Detected Not Covered 4 55953816 p.V1199fs*27 DEL − 5-15% Detected Detected Detected Not Covered 4 55955112 p.G1145R SNV − 5-15% Detected Detected Detected Not Covered 4 55961023 p.A973S SNV − 5-15% Detected Detected Detected Not Covered 4 55962505 p.G873G SNV − 5-15% Detected Detected Detected Not Covered 4 55962545 p.(=) INS − genomic Not Covered Detected Detected Not Covered 4 55972974 p.Q472H SNV − genomic Not Covered Detected Detected Not Covered 4 55972978 p.? DEL − 5-15% Detected Detected Not Detected Not Covered 4 55979623 p.R275L SNV − 5-15% Detected Detected Detected Not Covered 4 55980239 p.(=) SNV − genomic Not Covered Detected Not Covered Not Covered 4 153244078 p.E693E SNV − 5-15% Detected Not Covered Not Covered Detected 4 153244092 p.R689W SNV − 5-15% Detected Not Covered Not Covered Detected 4 153244124 p.S678* SNV − 5-15% Detected Not Covered Not Covered Detected 4 153244155 p.S668fs*39 DEL − 5-15% Detected Not Covered Not Covered Detected 4 153245477 p.N492fs*42 INS − 5-15% Detected Not Detected Detected Detected 4 153247222 p.D527G SNV − 5-15% Detected Not Covered Not Covered Detected 4 153247226 p.W526R SNV − 5-15% Detected Not Covered Not Covered Detected 4 153247244 p.D520N SNV − 5-15% Detected Not Covered Not Covered Detected 4 153247351 p.R484M SNV − 5-15% Detected Detected Detected Detected 4 153247366 p.R479Q SNV − 5-15% Detected Detected Detected Detected 4 153249384 p.R465H SNV − 5-15% Detected Detected Detected Detected 4 153249440 p.W446* SNV − 5-15% Detected Detected Detected Detected 4 153249456 p.R441L SNV − 5-15% Detected Detected Detected Detected 4 153250883 p.R393* SNV − 5-15% Detected Detected Detected Detected 4 153258983 p.R278* SNV − 5-15% Detected Detected Detected Not Covered 4 153259071 p.E248D SNV − 5-15% Detected Not Covered Detected Not Covered 5 112173830 p.D849fs*2 INS + 15-35%  Detected Not Covered Not Covered Detected 5 112173917 p.R876* SNV + 15-35%  Detected Detected Detected Detected 5 112173930 p.I880T SNV + 15-35%  Detected Detected Detected Detected 5 112173947 p.Q886* SNV + 15-35%  Detected Detected Detected Detected 5 112174043 p.E918* SNV + 15-35%  Detected Not Covered Detected Not Covered 5 112174577 p.Q1096* SNV + 15-35%  Detected Detected Not Covered Detected 5 112174596 p.Y1102C SNV + 15-35%  Detected Detected Not Covered Detected 5 112174726 p.E1145E SNV + 15-35%  Detected Not Covered Detected Detected 5 112174989 p.S1234fs*31 DEL + 15-35%  Detected Not Covered Not Covered Detected 5 112175086 p.E1265E SNV + 15-35%  Detected Not Covered Detected Detected 5 112175162 p.Q1291* SNV + 15-35%  Detected Detected Detected Detected 5 112175171 p.Q1294* SNV + 15-35%  Detected Detected Detected Detected 5 112175211 p.E1309fs*6 INS + 15-35%  Detected Detected Detected Detected 5 112175255 p.E1322* SNV + 15-35%  Detected Detected Detected Detected 5 112175348 p.E1353* SNV + 15-35%  Detected Detected Detected Detected 5 112175354 p.S1355P SNV + 15-35%  Detected Detected Detected Detected 5 112175423 p.Q1378* SNV + 15-35%  Detected Detected Detected Not Covered 5 112175432 p.P1381S SNV + 15-35%  Detected Detected Detected Not Covered 5 112175479 p.R1399fs*9 DEL + 15-35%  Detected Not Covered Detected Detected 5 112175507 p.Q1406* SNV + 15-35%  Detected Not Covered Detected Detected 5 112175576 p.Q1429* SNV + 15-35%  Detected Detected Detected Detected 5 112175675 p.S1465fs*3 DEL + 15-35%  Detected Detected Detected Detected 5 112175830 p.P1514fs*9 DEL + 15-35%  Detected Detected Detected Detected 5 112175852 p.E1521* SNV + 15-35%  Detected Detected Detected Detected 5 112175930 p.E1547* SNV + 15-35%  Detected Detected Detected Detected 5 112175945 p.E1552* SNV + 15-35%  Detected Detected Detected Detected 5 112175951 p.T1556fs*3 INS + 15-35%  Detected Detected Detected Detected 5 112176063 p.P1594fs*38 INS + 15-35%  Detected Not Covered Not Covered Detected 5 112176117 p.P1609L SNV + 15-35%  Detected Not Covered Not Covered Detected 5 149433596 p.(=) SNV − genomic Not Covered Detected Not Covered Not Covered 5 149433597 p.(=) SNV − genomic Not Covered Detected Not Covered Not Covered 5 149433645 p.Y969C SNV − 15-35%  Detected Detected Detected Not Covered 5 149433673 p.A960T SNV − 15-35%  Detected Detected Detected Not Covered 5 149453057 p.? INS − 15-35%  Detected Detected Detected Not Covered 5 170837543 p.W288fs*12 INS + 15-35%  Detected Detected Detected Not Covered 7 55211080 p.R108K SNV + 15-35%  Detected Detected Detected Not Covered 7 55211097 p.E114K SNV + 15-35%  Detected Detected Detected Not Covered 7 55211165 p.P136P SNV + 15-35%  Detected Not Covered Detected Not Covered 7 55221822 p.A289V SNV + 15-35%  Detected Detected Detected Not Covered 7 55221830 p.V292L SNV + 15-35%  Detected Detected Detected Not Covered 7 55233043 p.G598V SNV + 15-35%  Detected Detected Detected Not Covered 7 55233109 p.C620Y SNV + 15-35%  Detected Not Covered Detected Not Covered 7 55241615 p.L688P SNV + 15-35%  Detected Not Covered Not Covered Detected 7 55241644 p.A698T SNV + 15-35%  Detected Detected Not Covered Detected 7 55241708 p.G719A SNV + 15-35%  Detected Detected Detected Detected 7 55241722 p.G724S SNV + 15-35%  Detected Detected Detected Detected 7 55241755 p.(=) SNV + genomic Not Covered Not Covered Detected Not Detected 7 55242427 p.P733S SNV + 15-35%  Detected Detected Detected Detected 7 55242433 p.G735S SNV + 15-35%  Detected Detected Detected Detected 7 55242452 p.P741L SNV + 15-35%  Detected Detected Detected Detected 7 55242464 p.E746_A750delELREA DEL + 15-35%  Detected Detected Detected Detected 7 55248995 p.V765M SNV + 15-35%  Detected Detected Detected Detected 7 55249063 p.Q787Q SNV + genomic Not Covered Detected Detected Detected 7 55249077 p.L792P SNV + 15-35%  Detected Detected Detected Detected 7 55249131 p.G810D SNV + 15-35%  Detected Detected Detected Detected 7 55249143 p.L814P SNV + 15-35%  Detected Detected Detected Detected 7 55259427 p.E829K SNV + 15-35%  Detected Not Covered Not Covered Detected 7 55259439 p.L833V SNV + 15-35%  Detected Not Covered Not Covered Detected 7 55259446 p.H835L SNV + 15-35%  Detected Not Covered Not Covered Detected 7 55259457 p.A839T SNV + 15-35%  Detected Not Covered Not Covered Detected 7 55259515 p.L858R SNV + 15-35%  Detected Detected Detected Detected 7 55259524 p.L861Q SNV + 15-35%  Detected Detected Detected Detected 7 55259530 p.G863D SNV + 15-35%  Detected Detected Detected Detected 7 55259554 p.A871G SNV + 15-35%  Detected Detected Detected Detected 7 116339642 p.E168D SNV + 15-35%  Detected Detected Detected Detected 7 116340262 p.N375S SNV + 15-35%  Detected Detected Detected Detected 7 116412044 p.? SNV + 15-35%  Detected Not Covered Detected Detected 7 116417465 p.H1112H SNV + 15-35%  Detected Detected Detected Detected 7 116417499 p.H1124D SNV + 15-35%  Detected Detected Detected Detected 7 116418969 p.M1178I SNV + 15-35%  Detected Not Covered Not Covered Detected 7 116418997 p.R1188* SNV + 15-35%  Detected Not Covered Not Covered Detected 7 116419008 p.T1191T SNV + 15-35%  Detected Not Covered Not Covered Detected 7 116422133 p.L1223W SNV + 15-35%  Detected Not Covered Not Covered Detected 7 116423428 p.Y1253D SNV + 15-35%  Detected Detected Detected Detected 7 116423449 p.G1260C SNV + 15-35%  Detected Detected Detected Detected 7 116423456 p.K1262R SNV + 15-35%  Detected Detected Detected Detected 7 116423474 p.M1268T SNV + 15-35%  Detected Detected Detected Detected 7 116436022 p.A1357A SNV + genomic Not Covered Not Covered Not Covered Detected 7 116436097 p.P1382P SNV + genomic Not Covered Not Covered Not Covered Detected 7 128845101 p.R199W SNV + 15-35%  Detected Detected Detected Not Covered 7 128846040 p.A324T SNV + 15-35%  Detected Detected Detected Not Covered 7 128846398 p.L412F SNV + 15-35%  Detected Detected Detected Not Covered 7 128846469 p.(=) SNV + genomic Not Covered Not Covered Detected Not Covered 7 128850341 p.W535L SNV + 15-35%  Detected Detected Detected Not Covered 7 128851593 p.T640A SNV + 15-35%  Detected Detected Detected Not Covered 7 140453136 p.V600E SNV − 15-35%  Detected Detected Detected Detected 7 140453145 p.L597R SNV − 15-35%  Detected Detected Detected Detected 7 140453154 p.D594G SNV − 15-35%  Detected Detected Detected Detected 7 140453193 p.N581S SNV − 15-35%  Detected Detected Detected Detected 7 140481417 p.G464V SNV − 15-35%  Detected Detected Detected Detected 7 140481428 p.G460G SNV − 15-35%  Detected Detected Detected Detected 7 140481449 p.P453P SNV − 15-35%  Detected Detected Detected Detected 7 140481478 p.R444W SNV − 15-35%  Detected Detected Not Covered Detected 7 148508727 p.Y646F SNV − 15-35%  Detected Detected Not Covered Not Covered 8 38282147 p.N272N SNV − 15-35%  Detected Detected Not Covered Not Covered 8 38285864 P150S SNV − 15-35%  Detected Detected Not Covered Not Covered 8 38285891 p.T141A SNV − 15-35%  Detected Detected Not Covered Not Covered 8 38285938 p.S125L SNV − 15-35%  Detected Detected Detected Not Covered 9 5073770 p.V617F SNV + 15-35%  Detected Detected Detected Not Covered 9 5073781 p.D620E SNV + 15-35%  Detected Detected Detected Not Covered 9 21971000 p.E120* SNV − 15-35%  Detected Detected Not Covered Not Covered 9 21971017 p.P114L SNV − 15-35%  Detected Detected Not Covered Not Covered 9 21971028 p.VV110* SNV − 15-35%  Detected Detected Not Covered Not Covered 9 21971036 p.D108Y SNV − 15-35%  Detected Detected Not Covered Not Covered 9 21971111 p.H83Y SNV − 15-35%  Detected Detected Not Covered Not Covered 9 21971120 p.R80* SNV − 15-35%  Detected Detected Not Covered Not Covered 9 21971153 p.E69* SNV − 15-35%  Detected Detected Not Detected Not Covered 9 21971186 p.R58* SNV − 15-35%  Detected Detected Detected Not Covered 9 80336317 p.T334T SNV − 15-35%  Detected Not Covered Detected Not Covered 9 80343489 p.D277fs*20 INS − 15-35%  Detected Not Covered Detected Detected 9 80343605 p.? DEL − 15-35%  Detected Not Covered Detected Not Detected 9 80409345 p.(=) SNV − genomic Not Covered Not Covered Detected Not Covered 9 80409432 p.M227_F228 > SI MNV − 15-35%  Detected Detected Detected Detected 9 80409533 p.? DEL − 15-35%  Detected Not Covered Not Detected Not Detected 9 80412493 p.R183Q SNV − 15-35%  Detected Not Covered Detected Detected 9 80412518 p.T175S SNV − 15-35%  Detected Not Covered Detected Detected 9 133738342 p.L248V SNV + 15-35%  Detected Detected Detected Not Covered 9 133738349 p.G250E SNV + 15-35%  Detected Detected Detected Not Covered 9 133738357 p.Y253H SNV + 15-35%  Detected Detected Detected Not Covered 9 133738363 p.E255K SNV + 15-35%  Detected Detected Detected Not Covered 9 133747520 p.D276G SNV + 15-35%  Detected Detected Detected Not Covered 9 133747571 p.I293 > MP INS + 15-35%  Detected Not Covered Detected Not Covered 9 133748391 p.M351T SNV + 15-35%  Detected Detected Detected Not Covered 9 133748403 p.E355G SNV + 15-35%  Detected Detected Detected Not Covered 9 133748414 p.F359V SNV + 15-35%  Detected Detected Detected Not Covered 9 133750319 p.L384M SNV + 15-35%  Detected Detected Not Covered Not Covered 9 133750356 p.H396R SNV + 15-35%  Detected Detected Detected Not Covered 9 139390779 p.S2471* SNV − 15-35%  Detected Detected Not Covered Not Covered 9 139390804 p.A2463fs*14 DEL − 15-35%  Detected Not Detected Not Covered Not Covered 9 139390816 p.Q2459* SNV − 15-35%  Detected Detected Not Covered Not Covered 9 139390873 p.Q2440* SNV − 15-35%  Detected Detected Not Covered Not Covered 9 139397768 p.L1678P SNV − 15-35%  Detected Detected Detected Not Covered 9 139397776 p.I1675I SNV − 15-35%  Detected Detected Detected Not Covered 9 139399344 p.L1600P SNV − 15-35%  Detected Detected Detected Not Covered 9 139399350 p.R1598P SNV − 15-35%  Detected Detected Detected Not Covered 9 139399365 p.L1593P SNV − 15-35%  Detected Detected Detected Not Covered 9 139399422 p.L1574P SNV − 15-35%  Detected Detected Detected Not Covered 10 43609096 p.C618R SNV + 15-35%  Detected Detected Detected Not Covered 10 43609102 p.C620R SNV + 15-35%  Detected Detected Detected Not Covered 10 43609942 p.E632_T636 > SS Com- + 15-35%  Detected Detected Detected Not Covered plex 10 43609990 p.V648I SNV + 15-35%  Detected Detected Detected Not Covered 10 43610039 p.A664D SNV + 15-35%  Detected Not Covered Detected Not Covered 10 43613840 p.E768D SNV + 15-35%  Detected Detected Detected Not Covered 10 43613843 p.L769L SNV + genomic Not Covered Detected Detected Not Covered 10 43615568 p.A883F MNV + 15-35%  Detected Detected Detected Not Covered 10 43615622 p.E901K SNV + 15-35%  Detected Detected Detected Not Covered 10 43617416 p.M918T SNV + 15-35%  Detected Detected Detected Not Covered 10 89624245 p.E7* SNV + 15-35%  Detected Detected Detected Detected 10 89624266 p.R14G SNV + 15-35%  Detected Detected Detected Detected 10 89624275 p.Q17* SNV + 15-35%  Detected Detected Detected Detected 10 89624297 p.D24G SNV + 15-35%  Detected Detected Detected Detected 10 89653686 p.(=) SNV + genomic Not Covered Not Covered Not Covered Detected 10 89653782 p.Y27C SNV + 15-35%  Detected Not Covered Not Covered Detected 10 89653814 p.P38S SNV + 15-35%  Detected Not Covered Not Covered Detected 10 89653844 p.N48D SNV + 15-35%  Detected Not Covered Not Covered Detected 10 89653858 p.D52D SNV + 15-35%  Detected Not Covered Not Covered Detected 10 89653865 p.R55G SNV + 15-35%  Detected Not Covered Not Covered Detected 10 89685271 p.F56V SNV + 15-35%  Detected Detected Not Covered Detected 10 89685307 p.Y68H SNV + 15-35%  Detected Detected Detected Detected 10 89685319 p.? SNV + 15-35%  Detected Detected Detected Detected 10 89690805 p.C71Y SNV + 15-35%  Detected Not Covered Not Covered Detected 10 89690818 p.Y76fs*1 DEL + 15-35%  Detected Not Covered Not Covered Detected 10 89690838 p.N82T SNV + 15-35%  Detected Not Covered Not Covered Detected 10 89690847 p.? SNV + 15-35%  Detected Not Covered Not Covered Detected 10 89692779 p.Y88C SNV + 15-35%  Detected Not Covered Not Covered Detected 10 89692818 p.I101T SNV + 15-35%  Detected Detected Not Covered Detected 10 89692830 p.C105F SNV + 15-35%  Detected Detected Not Covered Detected 10 89692850 p.L112V SNV + 15-35%  Detected Detected Not Covered Detected 10 89692911 p.G132D SNV + 15-35%  Detected Detected Not Covered Detected 10 89692965 p.E150G SNV + 15-35%  Detected Not Covered Not Covered Detected 10 89692980 p.Y155C SNV + 15-35%  Detected Not Covered Not Covered Detected 10 89692993 p.R159S SNV + 15-35%  Detected Not Covered Not Covered Detected 10 89711855 p.? DEL + 15-35%  Detected Detected Not Covered Not Detected 10 89711960 p.L193P SNV + 15-35%  Detected Not Covered Detected Detected 10 89711972 p.M199del DEL + 15-35%  Detected Not Covered Detected Detected 10 89711992 p.P204T SNV + 15-35%  Detected Not Covered Detected Detected 10 89711997 p.M205I SNV + 15-35%  Detected Not Covered Detected Detected 10 89717672 p.R233* SNV + 15-35%  Detected Detected Detected Detected 10 89717678 p.E235* SNV + 15-35%  Detected Detected Detected Detected 10 89717696 p.F241L SNV + 15-35%  Detected Detected Detected Detected 10 89717762 p.K263* SNV + 15-35%  Detected Detected Detected Detected 10 89717769 p.K267fs*9 DEL + 15-35%  Detected Detected Detected Detected 10 89720728 p.G293G SNV + 15-35%  Detected Detected Detected Not Covered 10 89720744 p.E299* SNV + 15-35%  Detected Detected Detected Not Covered 10 89720798 p.T319fs*1 DEL + 15-35%  Detected Detected Detected Not Covered 10 89720874 p.K342R SNV + 15-35%  Detected Detected Detected Not Covered 10 89720907 p.(=) SNV + genomic Not Covered Not Covered Detected Not Covered 10 89725042 p.? SNV + 15-35%  Detected Not Covered Not Covered Detected 10 89725056 p.F347fs*13 DEL + 15-35%  Detected Not Covered Not Covered Detected 10 89725072 p.E352G SNV + 15-35%  Detected Not Covered Not Covered Detected 10 89725108 p.S364C SNV + 15-35%  Detected Not Covered Not Covered Detected 10 123258034 p.N549K SNV − 5-15% Detected Detected Detected Not Covered 10 123274774 p.C382R SNV − 5-15% Detected Detected Detected Not Covered 10 123274794 p.Y375C SNV − 5-15% Detected Detected Detected Not Covered 10 123274810 p.T370A SNV − 5-15% Detected Detected Detected Not Covered 10 123279503 p.K310R SNV − 5-15% Detected Detected Detected Detected 10 123279519 p.G305R SNV − 5-15% Detected Detected Detected Detected 10 123279677 p.S252W SNV − 5-15% Detected Detected Detected Detected 11 533874 p.Q61R SNV − 5-15% Detected Detected Detected Not Covered 11 533881 p.A59T SNV − 5-15% Detected Detected Detected Not Covered 11 534242 p.H27H SNV − genomic Not Covered Detected Not Covered Not Covered 11 534288 p.G12V SNV − 5-15% Detected Detected Detected Not Covered 11 108117798 p.R337C SNV + 5-15% Detected Detected Not Covered Not Covered 11 108119823 p.V410A SNV + 5-15% Detected Detected Detected Not Covered 11 108123551 p.P604S SNV + 5-15% Detected Detected Detected Not Covered 11 108123641 p.? SNV + 5-15% Detected Not Covered Detected Not Covered 11 108138003 p.F858L SNV + 5-15% Detected Detected Detected Not Covered 11 108155132 p.A1309T SNV + 5-15% Detected Detected Not Detected Not Covered 11 108170479 p.D1682Y SNV + 5-15% Detected Detected Detected Not Covered 11 108170587 p.L1718V SNV + 5-15% Detected Not Covered Detected Not Covered 11 108172374 p.? SNV + 5-15% Detected Detected Not Covered Not Covered 11 108172385 p.R1730* SNV + 5-15% Detected Detected Not Covered Not Covered 11 108172421 p.A1742P SNV + 5-15% Detected Detected Detected Not Covered 11 108173640 p.L1794L SNV + 5-15% Detected Detected Detected Not Covered 11 108173736 p.L1826V SNV + 5-15% Detected Not Covered Detected Not Covered 11 108180945 p.V1941L SNV + 5-15% Detected Detected Detected Not Covered 11 108200958 p.Q2442P SNV + 5-15% Detected Detected Detected Not Covered 11 108204681 p.T2666A SNV + 5-15% Detected Detected Detected Not Covered 11 108205769 p.G2695A SNV + 5-15% Detected Detected Detected Not Covered 11 108205780 p.P2699T SNV + 5-15% Detected Detected Detected Not Covered 11 108206594 p.D2725V SNV + 5-15% Detected Detected Detected Not Covered 11 108218045 p.N2875S SNV + 5-15% Detected Detected Not Covered Not Covered 11 108218089 p.L2890V SNV + 5-15% Detected Detected Detected Not Covered 11 108218196 p.(=) SNV + genomic Not Covered Not Covered Detected Not Covered 11 108225590 p.T2947S SNV + 5-15% Detected Detected Detected Not Covered 11 108225661 p.(=) SNV + genomic Not Covered Not Covered Detected Not Covered 11 108236087 p.R3008H SNV + 5-15% Detected Detected Detected Not Covered 11 108236118 p.K3018K SNV + 5-15% Detected Detected Detected Not Covered 11 108236203 p.R3047* SNV + 5-15% Detected Detected Detected Not Covered 12 25362805 p.R164Q SNV − 5-15% Detected Not Covered Not Covered Detected 12 25378647 p.K117N SNV − 5-15% Detected Detected Detected Detected 12 25380275 p.Q61H SNV − 5-15% Detected Detected Detected Detected 12 25380283 p.A59T SNV − 5-15% Detected Detected Detected Detected 12 25398207 p.? SNV − 5-15% Detected Detected Not Covered Detected 12 25398215 p.T35I SNV − 5-15% Detected Detected Not Covered Detected 12 25398284 p.G12D SNV − 5-15% Detected Detected Detected Detected 12 25398295 p.V8V SNV − 5-15% Detected Detected Detected Detected 12 112888165 p.D61Y SNV + 5-15% Detected Detected Detected Not Covered 12 112888189 p.E69K SNV + 5-15% Detected Detected Detected Not Covered 12 112888199 p.A72V SNV + 5-15% Detected Detected Detected Not Covered 12 112888210 p.E76K SNV + 5-15% Detected Detected Detected Not Covered 12 112926852 p.P491L SNV + 5-15% Detected Detected Not Covered Not Covered 12 112926888 p.G503A SNV + 5-15% Detected Detected Detected Not Covered 12 112926899 p.T507A SNV + 5-15% Detected Detected Detected Not Covered 12 112926908 p.Q510K SNV + 5-15% Detected Detected Detected Not Covered 12 121431413 p.W206L SNV + 5-15% Detected Detected Detected Not Covered 12 121431428 p.Q211P SNV + 5-15% Detected Detected Detected Not Covered 12 121431481 p.R229* SNV + 5-15% Detected Not Covered Detected Not Covered 12 121431506 p.N237S SNV + 5-15% Detected Not Covered Detected Not Covered 12 121432032 p.T260M SNV + 5-15% Detected Detected Not Covered Not Covered 12 121432040 p.R263C SNV + 5-15% Detected Detected Not Covered Not Covered 12 121432117 p.G292fs*25 INS + 5-15% Detected Not Covered Detected Not Covered 12 121432117 p.G288G SNV + genomic Not Covered Not Covered Detected Not Covered 13 28592629 p.D839G SNV − 5-15% Detected Detected Detected Not Covered 13 28592642 p.D835Y SNV − 5-15% Detected Detected Detected Not Covered 13 28592653 p.G831E SNV − 5-15% Detected Detected Detected Not Covered 13 28602329 p.A680V SNV − 5-15% Detected Detected Detected Not Covered 13 28608255 p.D600_L601insFREYEYD INS − 5-15% Detected Not Detected Not Detected Not Covered 13 28608281 p.V592A SNV − 5-15% Detected Detected Detected Not Covered 13 28610138 p.S451F SNV − 5-15% Detected Detected Detected Not Covered 13 28610183 p.? SNV − genomic Not Covered Detected Detected Not Covered 13 48919244 p.E137* SNV + 5-15% Detected Detected Detected Not Covered 13 48923148 p.L199* SNV + 5-15% Detected Detected Detected Not Covered 13 48941628 p.? SNV + 5-15% Detected Detected Not Covered Not Covered 13 48941648 p.R320* SNV + 5-15% Detected Detected Not Covered Not Covered 13 48941658 p.E323G SNV + 5-15% Detected Detected Not Covered Not Covered 13 48941672 p.N328D SNV + 5-15% Detected Detected Not Covered Not Covered 13 48942685 p.R358* SNV + 5-15% Detected Detected Detected Not Covered 13 48953760 p.R455* SNV + 5-15% Detected Detected Not Covered Not Covered 13 48955538 p.R552* SNV + 5-15% Detected Detected Detected Not Covered 13 48955550 p.R556* SNV + 5-15% Detected Detected Detected Not Covered 13 48955571 p.W563R SNV + 5-15% Detected Detected Detected Not Covered 13 49027168 p.R579* SNV + 5-15% Detected Detected Detected Not Covered 13 49027249 p.? SNV + 5-15% Detected Not Covered Detected Not Covered 13 49033890 p.L676fs*16 DEL + 5-15% Detected Detected Detected Not Covered 13 49033916 p.Q685* SNV + 5-15% Detected Detected Detected Not Covered 13 49033926 p.L688P SNV + 5-15% Detected Detected Detected Not Covered 13 49037865 p.? SNV + 5-15% Detected Detected Detected Not Covered 13 49037877 p.C706F SNV + 5-15% Detected Detected Detected Not Covered 13 49037903 p.K715* SNV + 5-15% Detected Detected Detected Not Covered 13 49037913 p.D718G SNV + 5-15% Detected Detected Detected Not Covered 13 49039164 p.E748* SNV + 5-15% Detected Detected Detected Not Covered 13 49039183 p.V754G SNV + 5-15% Detected Detected Detected Not Covered 13 49039189 p.Y756C SNV + 5-15% Detected Detected Detected Not Covered 13 49039215 p.K765* SNV + 5-15% Detected Detected Detected Not Covered 14 105246551 p.E17K SNV − 5-15% Detected Detected Detected Detected 15 66727455 p.K57N SNV + 5-15% Detected Not Covered Not Covered Detected 15 66727483 p.D67N SNV + 5-15% Detected Not Covered Not Covered Detected 15 90631838 p.R172K SNV − 5-15% Detected Detected Not Covered Not Covered 15 90631879 p.P158P SNV − 5-15% Detected Detected Not Covered Not Covered 15 90631934 p.R140Q SNV − 5-15% Detected Detected Not Covered Not Covered 16 68835650 p.G81_F91del DEL + 5-15% Detected Not Detected Not Detected Not Covered 16 68846087 p.E353G SNV + 5-15% Detected Detected Not Covered Detected 16 68846137 p.D370H SNV + 5-15% Detected Detected Detected Detected 16 68847282 p.D402N SNV + 5-15% Detected Detected Detected Detected 16 68855924 p.G579fs*9 INS + 5-15% Detected Not Covered Not Covered Detected 16 68855934 p.L581P SNV + 5-15% Detected Not Covered Not Covered Detected 16 68855966 p.A592T SNV + 5-15% Detected Not Covered Not Covered Detected 16 68856041 p.A617T SNV + 5-15% Detected Not Covered Not Covered Detected 16 68856093 p.A634V SNV + 5-15% Detected Not Covered Not Covered Detected 16 68856105 p.W638* SNV + 5-15% Detected Not Covered Not Covered Detected 17 7572962 p.K382fs* > 12 DEL − 5-15% Detected Not Covered Not Covered Detected 17 7572986 p.Q375* SNV − 5-15% Detected Not Covered Not Covered Detected 17 7573010 p.? SNV − 5-15% Detected Not Covered Not Covered Detected 17 7574003 p.R342* SNV − 5-15% Detected Detected Detected Detected 17 7574012 p.E339* SNV − 5-15% Detected Detected Detected Detected 17 7574018 p.R337C SNV − 5-15% Detected Detected Detected Detected 17 7574026 p.G334V SNV − 5-15% Detected Detected Detected Detected 17 7576855 p.Q331* SNV − 5-15% Detected Not Covered Not Covered Detected 17 7576865 p.Y327* SNV − 5-15% Detected Not Covered Not Covered Detected 17 7576883 p.K321K SNV − 5-15% Detected Not Covered Not Covered Detected 17 7576897 p.Q317* SNV − 5-15% Detected Not Covered Not Covered Detected 17 7577022 p.R306* SNV − 5-15% Detected Detected Detected Detected 17 7577046 p.E298* SNV − 5-15% Detected Detected Detected Detected 17 7577105 p.P278L SNV − 5-15% Detected Detected Detected Detected 17 7577120 p.R273H SNV − 5-15% Detected Detected Detected Detected 17 7577538 p.R248Q SNV − 5-15% Detected Detected Detected Detected 17 7577548 p.G245S SNV − 5-15% Detected Detected Detected Detected 17 7577559 p.S241F SNV − 5-15% Detected Detected Detected Detected 17 7577580 p.Y234C SNV − 5-15% Detected Detected Detected Detected 17 7578115 p.(=) SNV − genomic Not Covered Not Covered Detected Not Covered 17 7578190 p.Y220C SNV − 5-15% Detected Detected Detected Detected 17 7578196 p.V218E SNV − 5-15% Detected Detected Detected Detected 17 7578203 p.V216M SNV − 5-15% Detected Detected Detected Detected 17 7578235 p.Y205C SNV − 5-15% Detected Detected Detected Detected 17 7578388 p.R181H SNV − 5-15% Detected Detected Detected Detected 17 7578442 p.Y163C SNV − 5-15% Detected Detected Detected Detected 17 7578449 p.A161T SNV − 5-15% Detected Detected Detected Detected 17 7578461 p.V157F SNV − 5-15% Detected Detected Detected Detected 17 7578526 p.C135Y SNV − 5-15% Detected Detected Detected Detected 17 7578535 p.K132R SNV − 5-15% Detected Detected Detected Detected 17 7578542 p.L130V SNV − 5-15% Detected Detected Detected Detected 17 7578550 p.S127F SNV − 5-15% Detected Detected Detected Detected 17 7579295 p.? SNV − 5-15% Detected Not Covered Not Covered Not Detected 17 7579312 p.T125T SNV − 5-15% Detected Not Covered Not Covered Detected 17 7579358 p.R110L SNV − 5-15% Detected Detected Detected Detected 17 7579368 p.Y107D SNV − 5-15% Detected Detected Detected Detected 17 7579414 p.W91* SNV − 5-15% Detected Detected Detected Detected 17 7579442 p.P82L SNV − 5-15% Detected Detected Detected Detected 17 7579472 p.R72P SNV − genomic Not Covered Detected Detected Detected 17 7579521 p.E56* SNV − 5-15% Detected Not Covered Not Covered Detected 17 7579536 p.E51* SNV − 5-15% Detected Not Covered Not Covered Detected 17 7579553 p.L45P SNV − 5-15% Detected Not Covered Not Covered Detected 17 7579575 p.Q38* SNV − 5-15% Detected Not Covered Not Covered Detected 17 7579715 p.P27fs*17 DEL − 5-15% Detected Not Covered Not Covered Detected 17 7579801 p.(=) SNV − genomic Not Covered Not Covered Not Covered Detected 17 37880220 p.L755S SNV + 5-15% Detected Detected Detected Not Covered 17 37880261 p.D769Y SNV + 5-15% Detected Detected Detected Not Covered 17 37880981 p.A775_G776insYVMA INS + 5-15% Detected Detected Not Covered Detected 17 37881332 p.V842I SNV + 5-15% Detected Detected Not Detected Not Covered 17 37881378 p.N857S SNV + 5-15% Detected Detected Detected Not Covered 17 37881440 p.H878Y SNV + 5-15% Detected Detected Detected Not Covered 18 48575112 p.P102P SNV + 5-15% Detected Detected Not Covered Not Covered 18 48575183 p.V126A SNV + 5-15% Detected Detected Detected Not Covered 18 48575195 p.P130L SNV + 5-15% Detected Detected Detected Not Covered 18 48575209 p.R135* SNV + 5-15% Detected Detected Detected Not Covered 18 48575671 p.S144* SNV + 5-15% Detected Detected Not Covered Not Covered 18 48581198 p.G168* SNV + 5-15% Detected Detected Detected Not Covered 18 48581229 p.S178* SNV + 5-15% Detected Detected Detected Not Covered 18 48581243 p.Q183* SNV + 5-15% Detected Detected Detected Not Covered 18 48584560 p.Q245* SNV + 5-15% Detected Detected Detected Not Covered 18 48584593 p.Q256* SNV + 5-15% Detected Detected Detected Not Covered 18 48584602 p.T259fs*4 DEL + 5-15% Detected Detected Detected Not Covered 18 48586262 p.Q311* SNV + 5-15% Detected Detected Detected Not Covered 18 48586291 p.? SNV + 5-15% Detected Detected Detected Not Covered 18 48591838 p.Q334R SNV + 5-15% Detected Detected Detected Detected 18 48591847 p.E337G SNV + 5-15% Detected Detected Detected Detected 18 48591855 p.K340E SNV + 5-15% Detected Detected Detected Detected 18 48591865 p.S343* SNV + 5-15% Detected Detected Detected Detected 18 48593405 p.G386R SNV + 5-15% Detected Detected Detected Not Covered 18 48593465 p.A406T SNV + 5-15% Detected Detected Detected Not Covered 18 48593475 p.Q410fs*6 INS + 5-15% Detected Detected Detected Not Covered 18 48593495 p.R416G SNV + 5-15% Detected Detected Detected Not Covered 18 48603032 p.R445* SNV + 5-15% Detected Detected Detected Not Covered 18 48604682 p.R502G SNV + 5-15% Detected Detected Detected Detected 18 48604697 p.K507E SNV + 5-15% Detected Detected Detected Detected 18 48604754 p.E526* SNV + 5-15% Detected Detected Detected Detected 18 48604769 p.R531R SNV + 5-15% Detected Detected Detected Detected 19 1207076 p.E57fs*7 DEL + 5-15% Detected Detected Detected Detected 19 1220321 p.(=) SNV + genomic Not Covered Detected Not Covered Detected 19 1220371 p.? SNV + 5-15% Detected Detected Not Covered Detected 19 1220382 p.Q159* SNV + 5-15% Detected Detected Not Covered Detected 19 1220487 p.D194Y SNV + 5-15% Detected Detected Detected Not Covered 19 1220502 p.E199* SNV + 5-15% Detected Detected Detected Not Covered 19 1221293 p.Y272Y SNV + 5-15% Detected Detected Detected Detected 19 1221319 p.P281L SNV + 5-15% Detected Detected Detected Detected 19 1223125 p.F354L SNV + 5-15% Detected Detected Detected Detected 19 3115012 p.R183C SNV + 5-15% Detected Not Covered Detected Not Covered 19 3118942 p.Q209L SNV + 5-15% Detected Detected Detected Not Covered 19 3119239 p.T257T SNV + genomic Not Covered Not Covered Detected Not Covered 19 17945696 p.V722I SNV − 5-15% Detected Detected Detected Not Covered 19 17948009 p.A572V SNV − 5-15% Detected Detected Detected Not Covered 20 36031631 p.P487L SNV + 5-15% Detected Not Covered Not Covered Detected 20 57480494 p.Y163Y SNV + 5-15% Detected Not Covered Not Covered Detected 20 57484420 p.R201C SNV + 5-15% Detected Detected Detected Detected 20 57484596 p.Q227L SNV + 5-15% Detected Detected Not Covered Not Covered 22 24133967 p.R40* SNV + 5-15% Detected Detected Detected Not Covered 22 24133990 p.Y47* SNV + 5-15% Detected Detected Detected Not Covered 22 24134006 p.R53* SNV + 5-15% Detected Detected Detected Not Covered 22 24143240 p.R158* SNV + 5-15% Detected Detected Detected Not Covered 22 24145528 p.L191fs*26 INS + 5-15% Detected Not Detected Not Covered Not Covered 22 24145582 p.R201* SNV + 5-15% Detected Detected Detected Not Covered 22 24145588 p.A203T SNV + 5-15% Detected Detected Detected Not Covered 22 24145675 p.(=) SNV + genomic Not Covered Not Covered Detected Not Covered 22 24176353 p.P383fs DEL + 5-15% Detected Detected Detected Not Covered 

What is claimed is:
 1. A single nucleic acid molecule comprising at least one reference sequence and multiple variants of the at least one reference sequence, wherein the nucleotide sequences of the at least one reference sequence and each variant are defined, wherein the at least one reference sequence and variants are arranged within the single nucleic acid molecule as cassettes, wherein within each cassette the at least one reference sequence is operably linked to one or more of a restriction enzyme site(s), and/or hairpin-forming site(s), wherein each variant is at least 50% identical to the reference sequence, and wherein the single nucleic acid molecule comprises at least 15 variants.
 2. The single nucleic acid molecule of claim 1 wherein the at least one reference sequence and variants are releasable from the nucleic acid molecule using a restriction enzyme.
 3. The single nucleic acid molecule of claim 1 wherein the variants comprise at least one single nucleotide polymorphism (SNP), multiple nucleotide polymorphisms (MNP), insertion, deletion, copy number variation, gene fusion, duplication, inversion, repeat polymorphism, homopolymer of a reference sequence, and/or a nonhuman sequence.
 4. The single nucleic acid molecule of claim 1 comprising at least 30 variants.
 5. The single nucleic acid molecule of claim 1 wherein each variant is related to cancer, an inherited disease, or infectious disease.
 6. A composition comprising the single nucleic acid molecule of claim 1 wherein the single nucleic acid molecule is a plasmid.
 7. The composition of claim 6 further comprising a cell wherein the plasmid is transfected into and/or mixed with the cell.
 8. The composition of claim 7 wherein the composition is formalin-fixed, paraffin embedded (FFPE) to produce a FFPE sample.
 9. A frequency ladder comprising the single nucleic acid molecule of claim 1 wherein the nucleic acid molecule is diluted to produce a plurality of variants at different frequencies.
 10. The frequency ladder of claim 9, wherein the single nucleic acid molecule is diluted with at least one nucleic acid comprising the reference sequence.
 11. The single nucleic acid molecule of claim 1 wherein the variants comprise a subsequence of the at least one reference sequence.
 12. The single nucleic acid molecule of claim 1 wherein the at least one reference sequence is selected from MPL, NRAS, ALK, MSH6, IDH1, ERBB4, VHL, MLH1, CTNNB1, FOXL2, PIK3CA, FGFR3, PDGFRA, KIT, KDR, FBXW7, APC, CSF1R, NPM1, EGFR, MET, SMO, BRAF, EZH2, FGFR1, JAK2, CDKN2A, GNAQ, ABL1, NOTCH1, RET, PTEN, FGFR2, HRAS, ATM, KRAS, PTPN11, HNF1A, FLT3, RB1, AKT1, IDH2, CDH1, TP53, ERBB2, SMAD4, STK11, GNA11, JAK3, SRC, GNAS, and SMARCB1.
 13. A composition comprising a single nucleic acid molecule comprising at least one reference sequence and multiple nucleic acid species of the at least one reference sequence, wherein the nucleotide sequences of the at least one reference sequence and each species are defined, and wherein the nucleic acid sequence of each species differs from its neighbor species by a predetermined percentage of 10% or less, and wherein each species comprises a homopolymer sequence of at least 3 nucleotides.
 14. A composition comprising a single nucleic acid molecule comprising at least one reference sequence and multiple nucleic acid species of the at least one reference sequence, wherein the nucleotide sequences of the at least one reference sequence and each species are defined, and wherein the nucleic acid sequence of each species differs from its neighbor species by a predetermined percentage of 10% or less, and wherein each species possess a nucleic acid barcode.
 15. A formalin-fixed, paraffin embedded (FFPE) sample composition comprising a cell and a plasmid, where the plasmid is transfected into and/or mixed with the cell, and where the plasmid is a single nucleic acid molecule comprising at least one reference sequence and multiple variants of the at least one reference sequence, wherein the nucleotide sequences of the at least one reference sequence and each variant are defined, wherein the at least one reference sequence and variants are arranged within the single nucleic acid molecule as cassettes, wherein within each cassette the at least one reference sequence is operably linked to one or more of a restriction enzyme site(s), and/or hairpin-forming site(s), and wherein each variants is at least 50% identical to the reference sequence. 